Ungulates expressing exogenous IGF-I in their milk

ABSTRACT

The present invention relates to animals that express exogenous growth factors in their milk, and in particular to pigs that express exogenous IGF-I in their milk. The present invention also relates to methods for increasing piglet weight gain and intestinal lactase activity. The present invention thus provides a method of facilitating piglet development and decreasing piglet mortality.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/225,474, filed Aug. 15, 2000, which is incorporated by reference inits entirety to the extent not inconsistent with the disclosureherewith.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Contract No.96-35206-3850 awarded by the U.S. Department of Agriculture. TheGovernment has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to animals that express exogenous growthfactors in their milk, and in particular to pigs that express exogenousIGF-I in their milk. The present invention also relates to methods forincreasing piglet weight gain and intestinal lactase enzyme activity.

BACKGROUND OF THE INVENTION

The early neonatal period is the time of greatest animal loss for porkproducers. The 1991 USDA National Swine Survey on Morbidity/Mortalityand Heath Management of Swine in the U.S. estimated that overallpre-weaning mortality was 15% and that nearly all cases of morbidity andmortality occurred in piglets less than 7 days of age (USDA, 1991).Importantly, 58% of the cases of morbidity were reportedly due to scoursand 30% of the mortality was attributed to scours or starvation.

In recent years, pork producers have reduced lactation lengths in anattempt to maximize the number of piglets born per sow per year.Lactation periods of 10-14 days are common in the swine industry. Thisproduction system creates a need for sows that produce high levels ofmilk in early lactation in order to obtain maximal piglet growth. Inaddition, the number of piglets born per litter has increased, thusadding to the demand for higher milk production early in lactation.

Low milk production is manifested by slow piglet growth before weaningand suboptimal growth post-weaning (Hartman et al., Symp. Zool. Sci.,51:301 [1984]). Milk production accounts for 44% of the growth weight ofthe piglets (Lewis et al., J. Anim. Sci., 47:634 [1978]). In addition,gastrointestinal disease in piglets reduces their survival. Suchdiseases are typically treated with antibiotics. It has also beensuggested that bioactive substances in milk may have important functionsin piglet growth and health.

Clearly, the industry would benefit from a method of increasing milkproduction and nutrient value in sow milk. Supplementing milk withgrowth factors or nutrients is too costly and labor-intensive to be aviable solution. The art is in need of a cost effective method ofincreasing milk production and nutrient value in lactating sows.

SUMMARY OF THE INVENTION

The present invention relates to animals that express exogenous growthfactors in their milk, and in particular to pigs that express exogenousIGF-I in their milk. The present invention also relates to methods forincreasing piglet weight gain and intestinal lactase enzyme activity.

In some embodiments, the present invention provides a transgenic animalhaving a genome comprising a heterologous nucleic acid sequence encodinga growth factor operably linked to a mammary preferential promoter,wherein descendants of the transgenic animal express an increased amountof growth factor in their milk as compared to control non-transgenicanimals. The present invention is not limited to any particular growthfactor or source of the nucleic acid encoding the growth factor. Indeed,a variety of growth factors are contemplated, including, but not limitedto insulin-like growth factor I, insulin-like growth factor II,epidermal growth factor, platelet derived growth factor, fibroblastgrowth factor, and transforming growth factor. The present invention isnot limited to any particular transgenic animal. Indeed, a variety oftransgenic animals are contemplated, including, but not limited toungulates such as pigs, cattle, sheep, and goats. The animal may benonhuman. The present invention is not limited to any particular genderof transgenic animal. Indeed, both male and female transgenic animalsare contemplated. The present invention is not limited to any particularIGF-I gene. Indeed, a variety of IGF-I genes are contemplated,including, but not limited to human, porcine, and bovine insulin-likegrowth factor I genes. In some particularly preferred embodiments, theinsulin-like growth factor I comprises SEQ ID NO:2. In other preferredembodiments, the heterologous nucleic acid sequence is encoded by SEQ IDNO:1. The present invention is not limited to any particular mammarypreferential promoter or source of the nucleic acid encoding thepromoter. Indeed, a variety of mammary preferential promoters arecontemplated, including, but not limited to alpha-lactalbumin promoters,whey acidic protein promoters, and casein promoters. In someembodiments, the gametes of said transgenic animal comprise saidheterologous nucleic acid sequence. When the animal is a heterozygote,it is understood that only a portion of the gametes will comprise thetransgene.

In other embodiments, the present invention provides compositionscomprising milk from a transgenic animal having a genome comprising aheterologous nucleic acid sequence encoding a growth factor operablylinked to a mammary preferential promoter, wherein said milk comprisesan increased amount of growth factor as compared to milk from controlnon-transgenic animals. The present invention is not limited to anyparticular growth factor or the particular source of the nucleic acidsequence encoding the growth factor. Indeed, a variety of growth factorsare contemplated, including, but not limited to insulin-like growthfactor I, insulin-like growth factor II, epidermal growth factor,platelet derived growth factor, fibroblast growth factor, andtransforming growth factor. The present invention is not limited to anyparticular transgenic animal. Indeed, a variety of transgenic animalsare contemplated, including, but not limited to ungulates such as pigs,cattle, sheep, and goats. The animal may be non-human. The presentinvention is not limited to any particular gender of transgenic animal.Indeed, both male and female transgenic animals are contemplated. Thepresent invention is not limited to any particular IGF-I gene or theparticular source of the nucleic acid encoding the growth factor.Indeed, a variety of IGF-I genes are contemplated, including, but notlimited to human, porcine, and bovine insulin-like growth factor Igenes. In some particularly preferred embodiments, the insulin-likegrowth factor I comprises SEQ ID NO:2. In other preferred embodiments,the heterologous nucleic acid sequence is encoded by SEQ ID NO:1. Thepresent invention is not limited to any particular mammary preferentialpromoter or the particular source of the nucleic acid encoding thepromoter. Indeed, a variety of mammary preferential promoters arecontemplated, including, but not limited to alpha-lactalbumin promoters,whey acidic protein promoters, and casein promoters.

In still further embodiments, the present invention provides methods forincreasing weight gain in a suckling animal, comprising: a) providing i)a transgenic animal having a genome comprising a heterologous nucleicacid sequence encoding a growth factor gene operably linked to a mammarypreferential promoter, wherein the transgenic animal expresses anincreased amount of growth factor in its milk as compared to controlnon-transgenic animals; and ii) a suckling offspring of the transgenicanimal; and b) providing the suckling offspring milk of said transgenicanimal, wherein the suckling offspring has increased weight gainrelative to a suckling offspring provided milk of a non-transgenicanimal. The present invention is not limited any particular growthfactor or the particular source of the nucleic acid encoding the growthfactor. Indeed, a variety of growth factors are contemplated, including,but not limited to insulin-like growth factor I, insulin-like growthfactor II, epidermal growth factor, platelet-derived growth factor,fibroblast growth factor, and transforming growth factor. The presentinvention is not limited to any particular transgenic animal. Indeed, avariety of transgenic animals are contemplated, including, but notlimited to ungulates such as pigs, cattle, sheep, and goats. The animalmay be non-human. The present invention is not limited to any particulargender of transgenic animal. Indeed, both male and female transgenicanimals are contemplated. Likewise, in preferred embodiments, thesuckling animal can be a piglet, calf, lamb, or kid. The presentinvention is not limited to any particular IGF-I gene or the particularsource of the nucleic acid encoding the growth factor. Indeed, a varietyof IGF-I genes are contemplated, including, but not limited to human,porcine, and bovine insulin-like growth factor I genes. In someparticularly preferred embodiments, the insulin-like growth factor Icomprises SEQ ID NO:2. In other preferred embodiments, the heterologousnucleic acid sequence is encoded by SEQ ID NO:1. The present inventionis not limited to any particular mammary preferential promoter or theparticular source of the nucleic acid encoding the promoter. Indeed, avariety of mammary preferential promoters are contemplated, including,but not limited to alpha-lactalbumin promoters, whey acidic proteinpromoters, and casein promoters.

In still other embodiments, the present invention provides methods forincreasing intestinal lactase activity in a suckling animal, comprising:a) providing i) a transgenic animal having a genome comprising aheterologous nucleic acid sequence encoding a growth factor operablylinked to a mammary preferential promoter, wherein the transgenic animalexpresses an increased amount of growth factor in its milk as comparedto control non-transgenic animals; and ii) a suckling offspring of thetransgenic animal; and b) providing the suckling offspring milk of thetransgenic animal, wherein the suckling offspring has increasedintestinal lactase activity relative to a suckling offspring providedmilk of a non-transgenic animal. The present invention is not limitedany particular growth factor or the particular source of the nucleicacid encoding the growth factor. Indeed, a variety of growth factors arecontemplated, including, but not limited to insulin-like growth factorI, insulin-like growth factor II, epidermal growth factor,platelet-derived growth factor, fibroblast growth factor, andtransforming growth factor. The present invention is not limited to anyparticular transgenic animal. Indeed, a variety of transgenic animalsare contemplated, including, but not limited to ungulates such as pigs,cattle, sheep, and goats. The animal may be non-human. The presentinvention is not limited to any particular gender of transgenic animal.Indeed, both male and female transgenic animals are contemplated.Likewise, in preferred embodiments, the suckling animal can be a piglet,calf, lamb, or kid. The present invention is not limited to anyparticular IGF-I gene or the particular source of the nucleic acidencoding the growth factor. Indeed, a variety of IGF-I genes arecontemplated, including, but not limited to human, porcine, and bovineinsulin-like growth factor I genes. In some particularly preferredembodiments, the insulin-like growth factor I comprises SEQ ID NO:2. Inother preferred embodiments, the heterologous nucleic acid sequence isencoded by SEQ ID NO:1. The present invention is not limited to anyparticular mammary preferential promoter or the particular source of thenucleic acid encoding the promoter. Indeed, a variety of mammarypreferential promoters are contemplated, including, but not limited toalpha-lactalbumin promoters, whey acidic protein promoters, and caseinpromoters.

In some embodiments, the present invention provides methods forincreasing intestinal cell division in a suckling animal, comprising: a)providing i) a transgenic animal having a genome comprising aheterologous nucleic acid sequence encoding a growth factor operablylinked to a mammary preferential promoter, wherein the transgenic animalexpresses an increased amount of growth factor in its milk as comparedto control non-transgenic animals; and ii) a suckling offspring of thetransgenic animal; and b) providing the suckling offspring milk of thetransgenic animal, wherein the suckling offspring has increasedintestinal cell division relative to a suckling offspring provided milkof a non-transgenic animal. The present invention is not limited anyparticular growth factor or the particular source of the nucleic acidencoding the growth factor. Indeed, a variety of growth factors arecontemplated, including, but not limited to insulin-like growth factorI, insulin-like growth factor II, epidermal growth factor, plateletderived growth factor, fibroblast growth factor, and transforming growthfactor. The present invention is not limited to any particulartransgenic animal. Indeed, a variety of transgenic animals arecontemplated, including, but not limited to ungulates such as pigs,cattle, sheep, and goats. The animal may be non-human. The presentinvention is not limited to any particular gender of transgenic animal.Indeed, both male and female transgenic animals are contemplated.Likewise, in preferred embodiments, the suckling animal can be a piglet,calf, lamb, or kid. The present invention is not limited to anyparticular IGF-I gene. Indeed, a variety of IGF-I genes arecontemplated, including, but not limited to human, porcine, and bovineinsulin-like growth factor I genes. In some particularly preferredembodiments, the insulin-like growth factor I comprises SEQ ID NO:2. Inother preferred embodiments, the heterologous nucleic acid sequence isencoded by SEQ ID NO:1. The present invention is not limited to anyparticular mammary preferential promoter or the particular source of thenucleic acid encoding the growth factor. Indeed, a variety of mammarypreferential promoters are contemplated, including, but not limited toalpha-lactalbumin promoters, whey acidic protein promoters, and caseinpromoters.

In other embodiments, the present invention provides methods forincreasing intestinal villi length in a suckling animal, comprising: a)providing i) a transgenic animal having a genome, said genome comprisinga heterologous nucleic acid sequence encoding a growth factor operablylinked to a mammary preferential promoter, wherein the transgenic animalexpresses an increased amount of growth factor in its milk as comparedto control non-transgenic animals; and ii) a suckling offspring of thetransgenic animal; and b) providing the suckling offspring milk of saidtransgenic animal, wherein the suckling offspring has increasedintestinal villi length relative to a suckling offspring provided milkof a non-transgenic animal. The present invention is not limited anyparticular growth factor or the particular source of the nucleic acidencoding the growth factor. Indeed, a variety of growth factors arecontemplated, including, but not limited to insulin-like growth factorI, insulin-like growth factor II, epidermal growth factor, plateletderived growth factor, fibroblast growth factor, and transforming growthfactor. The present invention is not limited to any particulartransgenic animal. Indeed, a variety of transgenic animals arecontemplated, including, but not limited to ungulates such as pigs,cattle, sheep, and goats. The animal may be non-human. The presentinvention is not limited to any particular gender of transgenic animal.Indeed, both male and female transgenic animals are contemplated.Likewise, in preferred embodiments, the suckling animal can be a piglet,calf, lamb, or kid. The present invention is not limited to anyparticular IGF-I gene. Indeed, a variety of IGF-I genes arecontemplated, including, but not limited to human, porcine, and bovineinsulin-like growth factor I genes. In some particularly preferredembodiments, the insulin-like growth factor I comprises SEQ ID NO:2. Inother preferred embodiments, the heterologous nucleic acid sequence isencoded by SEQ ID NO:1. The present invention is not limited to anyparticular mammary preferential promoter or the particular source of thenucleic acid encoding the growth factor. Indeed, a variety of mammarypreferential promoters are contemplated, including, but not limited toalpha-lactalbumin promoters, whey acidic protein promoters, and caseinpromoters.

In some embodiments, the present invention provides methods forincreasing resistance to intestinal pathogens in a suckling animal,comprising: a) providing i) a transgenic animal having a genomecomprising a heterologous nucleic acid sequence encoding a growth factoroperably linked to a mammary preferential promoter, wherein thetransgenic animal express an increased amount of growth factor in theirmilk as compared to control non-transgenic animals; ii) a sucklingoffspring of the transgenic animal; and b) providing the sucklingoffspring milk of said transgenic animal, wherein the suckling offspringhas increased resistance to intestinal parasites relative to a sucklingoffspring provided milk of a non-transgenic animal. The presentinvention is not limited any particular growth factor or the particularsource of the nucleic acid encoding the growth factor. Indeed, a varietyof growth factors are contemplated, including, but not limited toinsulin-like growth factor I, insulin-like growth factor II, epidermalgrowth factor, platelet derived growth factor, fibroblast growth factor,and transforming growth factor. The present invention is not limited toany particular transgenic animal. Indeed, a variety of transgenicanimals are contemplated, including, but not limited to ungulates suchas pigs, cattle, sheep, and goats. The animal may be non-human. Thepresent invention is not limited to any particular gender of transgenicanimal. Indeed, both male and female transgenic animals arecontemplated. Likewise, in preferred embodiments, the suckling animalcan be a piglet, calf, lamb, or kid. The present invention is notlimited to any particular IGF-I gene. Indeed, a variety of IGF-I genesare contemplated, including, but not limited to human, porcine, andbovine insulin-like growth factor I genes. In some particularlypreferred embodiments, the insulin-like growth factor I comprises SEQ IDNO:2. In other preferred embodiments, the heterologous nucleic acidsequence is encoded by SEQ ID NO:1. The present invention is not limitedto any particular mammary preferential promoter or the particular sourceof the nucleic acid encoding the promoter. Indeed, a variety of mammarypreferential promoters are contemplated, including, but not limited toalpha-lactalbumin promoters, whey acidic protein promoters, and caseinpromoters. The present invention is not limited to resistance to anyparticular pathogen. Indeed, resistance to a variety of pathogens iscontemplated, including, but not limited to rotovirus, coronavirus, E.coli, and Salmonella.

In some embodiments, the present invention provides a transgenic animalhaving a genome comprising a nucleic acid sequence encoding a growthfactor and encoding alpha-lactalbumin operably linked to a mammarypreferential promoter, said animal expressing an increased amount ofgrowth factor and an increased amount of alpha-lactalbumin in its milkas compared to control non-transgenic animals.

In other embodiments, the present invention provides a transgenic animalhaving a genome comprising a nucleic acid sequence encoding a growthfactor and encoding alpha-lactalbumin operably linked to a mammarypreferential promoter, said animal expressing an increased amount ofgrowth factor in its milk and an increased milk volume as compared tocontrol non-transgenic animals.

In other embodiments, the present invention provides a method ofincreasing the volume of milk and the growth factor content of milk intransgenic animals, said method comprising: providing a transgenicanimal having a genome, said genome comprising a heterologous nucleicacid sequence encoding a growth factor gene and encodingalpha-lactalbumin operably linked to a mammary preferential promoter,wherein said transgenic animal expresses an increased amount of growthfactor in its milk and an increased milk volume as compared to controlnon-transgenic animals.

DESCRIPTION OF THE FIGURES

FIG. 1 shows lactase activity in various intestinal segments in pigletsfed 1.0 mg/L IGF-I for 14 days and control piglets.

FIG. 2 shows lactase activity in various segments of piglet intestine inresponse to varied levels of oral IGF-I.

FIG. 3 shows the nucleic acid sequences of SEQ ID NOs:1 and 2.

FIG. 4 shows milk production of transgenic sows expressing IGF-I andnon-transgenic control sows.

FIG. 5 shows IGF-I content in the milk of transgenic sows expressingIGF-I and non-transgenic control sows.

FIG. 6 shows the level of milk solids in the milk of transgenic sowsexpressing IGF-I and non-transgenic control sows.

FIG. 7 shows protein content in the milk of transgenic sows expressingIGF-I and non-transgenic control sows.

FIG. 8 shows the lactose content in the milk of transgenic sowsexpressing IGF-I and non-transgenic control sows.

FIG. 9 shows the fat content in the milk of transgenic sows expressingIGF-I and non-transgenic control sows.

FIG. 10 shows weight gain of piglets suckling transgenic sows expressingIGF-I and non-transgenic control sows.

FIG. 11 shows lactase activity in the intestines of piglets sucklingtransgenic sows expressing IGF-I and non-transgenic control sows.

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below.

As used herein, the term “host cell” refers to any eukaryotic cell(e.g., mammalian cells, avian cells, amphibian cells, plant cells, fishcells, and insect cells), whether located in vitro or in vivo.

As used herein, the term “cell culture” refers to any in vitro cultureof cells. Included within this term are continuous cell lines (e.g.,with an immortal phenotype), primary cell cultures, finite cell lines(e.g., non-transformed cells), and any other cell population maintainedin vitro, including oocytes and embryos.

As used herein, the term “vector” refers to any genetic element, such asa plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.,which is capable of replication when associated with the proper controlelements and which can transfer gene sequences between cells. Thus, theterm includes cloning and expression vehicles, as well as viral vectors.

As used herein, the term “integrated” refers to a vector that is stablyinserted into the genome (i.e., into a chromosome) of a host cell.

As used herein, the term “genome” refers to the genetic material (e.g.,chromosomes) of an organism.

As used herein the term “suckling offspring” refers to an animal that isnursing a female of its species. The term “suckling offspring” includesboth progeny of a female animal as well as sucklings that have beengrafted onto the female animal.

As used herein, the term “mammary preferential promoter” refers to apromoter that preferentially causes the expression of a gene in themammary gland. Different “mammary preferential promoters” can effectdifferent expression profiles to a gene (e.g., exhibiting differentexpression levels at different times during lactation). “Mammarypreferential promoters” include those that effect high expression levelsat different stages of lactation, including early lactation (e.g., thehuman alpha-lactalbumin promoter). Examples of mammary specificpromoters include, but are not limited to alpha-lactalbumin promoters,whey acidic protein promoters, alpha-, beta- and kappa-casein promoters,and lactoferrin promoters. The term “mammary preferential promoters”encompasses mammary preferential promoters from all mammalian species(e.g., human, mouse, bovine, porcine, and ovine mammary preferentialpromoters.) Furthermore, the term “mammary preferential promoter”encompasses variants of the wild-type promoters.

The term “nucleotide sequence of interest” refers to any nucleotidesequence (e.g., RNA or DNA), the manipulation of which may be deemeddesirable for any reason (e.g., treat disease, confer improvedqualities, etc.), by one of ordinary skill in the art. Such nucleotidesequences include, but are not limited to, coding sequences ofstructural genes (e.g., reporter genes, selection marker genes,oncogenes, drug resistance genes, growth factors, etc.), and non-codingregulatory sequences which do not encode an mRNA or protein product(e.g., promoter sequence, polyadenylation sequence, terminationsequence, enhancer sequence, etc.). A “nucleic acid sequence ofinterest” may be a derived from the host organism or cell or may be a“heterologous nucleic acid sequence.”

As used herein, the terms “heterologous nucleic acid sequence,”“heterologous gene” or “exogenous gene” refer to a nucleic acid sequence(e.g., a gene) that is not naturally present in a host organism or cell,or is artificially introduced into a host organism or cell.

As used herein, the term “protein of interest” refers to a proteinencoded by a nucleic acid of interest.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises coding sequences necessary for the production of apolypeptide or precursor (e.g., proinsulin). The polypeptide can beencoded by a full length coding sequence or by any portion of the codingsequence so long as the desired activity or functional properties (e.g.,enzymatic activity, ligand binding, signal transduction, etc.) of thefull-length or fragment are retained. The term also encompasses thecoding region of a structural gene and includes sequences locatedadjacent to the coding region on both the 5′ and 3′ ends for a distanceof about 1 kb or more on either end such that the gene corresponds tothe length of the full-length mRNA. The sequences that are located 5′ ofthe coding region and which are present on the mRNA are referred to as5′ untranslated sequences. The sequences that are located 3′ ordownstream of the coding region and which are present on the mRNA arereferred to as 3′ untranslated sequences. The term “gene” encompassesboth cDNA and genomic forms of a gene. A genomic form or clone of a genecontains the coding region interrupted with non-coding sequences termed“introns” or “intervening regions” or “intervening sequences.” Intronsare segments of a gene which are transcribed into nuclear RNA (hnRNA);introns may contain regulatory elements such as enhancers. Introns areremoved or “spliced out” from the nuclear or primary transcript; intronstherefore are absent in the messenger RNA (mRNA) transcript. The mRNAfunctions during translation to specify the sequence or order of aminoacids in a nascent polypeptide.

As used herein, the term “gene expression” refers to the process ofconverting genetic information encoded in a gene into RNA (e.g., mRNA,rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e., via theenzymatic action of an RNA polymerase), and for protein encoding genes,into protein through “translation” of mRNA. Gene expression can beregulated at many stages in the process. “Up-regulation” or “activation”refers to regulation that increases the production of gene expressionproducts (i.e., RNA or protein), while “down-regulation” or “repression”refers to regulation that decrease production. Molecules (e.g.,transcription factors) that are involved in up-regulation ordown-regulation are often called “activators” and “repressors,”respectively.

Where “amino acid sequence” is recited herein to refer to an amino acidsequence of a naturally occurring or synthetic protein molecule, “aminoacid sequence” and like terms, such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

As used herein, the terms “nucleic acid molecule encoding,” “nucleicacid sequence encoding,” “DNA sequence encoding,” “DNA encoding,” “RNAsequence encoding,” and “RNA encoding” refer to the order or sequence ofdeoxyribonucleotides or ribonucleotides along a strand ofdeoxyribonucleic acid or ribonucleic acid. The order of thesedeoxyribonucleotides or ribonucleotides determines the order of aminoacids along the polypeptide (protein) chain. The DNA or RNA sequencethus codes for the amino acid sequence.

As used herein, the term “variant,” when used in reference to a protein,refers to proteins encoded by partially homologous nucleic acids so thatthe amino acid sequence of the proteins varies. As used herein, the term“variant” encompasses proteins encoded by homologous genes havingconservative amino acid substituations, nonconservative amino acidsubstitutions, or both that do not result in a change in proteinfunction, as well as proteins encoded by homologous genes having aminoacid substitutions that cause decreased (e.g., null mutations) proteinfunction or increased protein function.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, for the sequence“A-G-T,” is complementary to the sequence “T-C-A.” Complementarity maybe “partial,” in which only some of the nucleic acids' bases are matchedaccording to the base pairing rules. Or, there may be “complete” or“total” complementarity between the nucleic acids. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions, aswell as detection methods that depend upon binding between nucleicacids.

The terms “homology” and “percent identity” when used in relation tonucleic acids refers to a degree of complementarity. There may bepartial homology (i.e., partial identity) or complete homology (i.e.,complete identity). A partially complementary sequence is one that atleast partially inhibits a completely complementary sequence fromhybridizing to a target nucleic acid sequence and is referred to usingthe functional term “substantially homologous.” The inhibition ofhybridization of the completely complementary sequence to the targetsequence may be examined using a hybridization assay (Southern orNorthern blot, solution hybridization and the like) under conditions oflow stringency. A substantially homologous sequence or probe (i.e., anoligonucleotide which is capable of hybridizing to anotheroligonucleotide of interest) will compete for and inhibit the binding(i.e., the hybridization) of a completely homologous sequence to atarget sequence under conditions of low stringency. This is not to saythat conditions of low stringency are such that non-specific binding ispermitted; low stringency conditions require that the binding of twosequences to one another be a specific (i.e., selective) interaction.The absence of non-specific binding may be tested by the use of a secondtarget which lacks even a partial degree of complementarity (e.g., lessthan about 30% identity); in the absence of non-specific binding theprobe will not hybridize to the second non-complementary target.

The art knows well that numerous equivalent conditions may be employedto comprise low stringency conditions; factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components (e.g., thepresence or absence of formamide, dextran sulfate, polyethylene glycol)are considered and the hybridization solution may be varied to generateconditions of low stringency hybridization different from, butequivalent to, the above listed conditions. In addition, the art knowsconditions that promote hybridization under conditions of highstringency (e.g., increasing the temperature of the hybridization and/orwash steps, the use of formamide in the hybridization solution, etc.).

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described above.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe that can hybridize(i.e., it is the complement of) the single-stranded nucleic acidsequence under conditions of low stringency as described above.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids. A single molecule that contains pairing of complementarynucleic acids within its structure is said to be “self-hybridized.”

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature” of a nucleic acid. The melting temperature is thetemperature at which a population of double-stranded nucleic acidmolecules becomes half dissociated into single strands. The equation forcalculating the T_(m) of nucleic acids is well known in the art. Asindicated by standard references, a simple estimate of the T_(m) valuemay be calculated by the equation: T_(m)=81.5+0.41(% G+C), when anucleic acid is in aqueous solution at 1 M NaCl (See e.g., Anderson andYoung, Quantitative Filter Hybridization, in Nucleic Acid Hybridization[1985]). Other references include more sophisticated computations thattake structural as well as sequence characteristics into account for thecalculation of T_(m).

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. With “high stringency” conditions, nucleicacid base pairing will occur only between nucleic acid fragments thathave a high frequency of complementary base sequences. Thus, conditionsof “weak” or “low” stringency are often required with nucleic acids thatare derived from organisms that are genetically diverse, as thefrequency of complementary sequences is usually less.

“High stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Medium stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH2PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 1.0×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Low stringency conditions” comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.1% SDS, 5×Denhardt's reagent [50×Denhardt's contains per 500ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and100 μg/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 5×SSPE, 0.1% SDS at 42° C. when a probe of about 500nucleotides in length is employed.

A gene may produce multiple RNA species that are generated bydifferential splicing of the primary RNA transcript. cDNAs that aresplice variants of the same gene will contain regions of sequenceidentity or complete homology (representing the presence of the sameexon or portion of the same exon on both cDNAs) and regions of completenon-identity (for example, representing the presence of exon “A” on cDNA1 wherein cDNA 2 contains exon “B” instead). Because the two cDNAscontain regions of sequence identity they will both hybridize to a probederived from the entire gene or portions of the gene containingsequences found on both cDNAs; the two splice variants are thereforesubstantially homologous to such a probe and to each other.

The terms “in operable combination,” “in operable order,” and “operablylinked” as used herein refer to the linkage of nucleic acid sequences insuch a manner that a nucleic acid molecule capable of directing thetranscription of a given gene and/or the synthesis of a desired proteinmolecule is produced. The term also refers to the linkage of amino acidsequences in such a manner so that a functional protein is produced.

As used herein, the term “selectable marker” refers to a gene thatencodes an enzymatic activity that confers the ability to grow in mediumlacking what would otherwise be an essential nutrient (e.g. the HIS3gene in yeast cells); in addition, a selectable marker may conferresistance to an antibiotic or drug upon the cell in which theselectable marker is expressed. Selectable markers may be “dominant”; adominant selectable marker encodes an enzymatic activity that can bedetected in any eukaryotic cell line. Examples of dominant selectablemarkers include the bacterial aminoglycoside 3′ phosphotransferase gene(also referred to as the neo gene) that confers resistance to the drugG418 in mammalian cells, the bacterial hygromycin G phosphotransferase(hyg) gene that confers resistance to the antibiotic hygromycin and thebacterial xanthine-guanine phosphoribosyl transferase gene (alsoreferred to as the gpt gene) that confers the ability to grow in thepresence of mycophenolic acid. Other selectable markers are not dominantin that their use must be in conjunction with a cell line that lacks therelevant enzyme activity. Examples of non-dominan selectable markersinclude the thymidine kinase (tk) gene that is used in conjunction withtk⁻ cell lines, the CAD gene which is used in conjunction withCAD-deficient cells and the mammalian hypoxanthine-guaninephosphoribosyl transferase (hprt) gene which is used in conjunction withhprt⁻ cell lines. A review of the use of selectable markers in mammaliancell lines is provided in Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, NewYork (1989) pp.16.9-16.15.

As used herein, the term “regulatory element” refers to a geneticelement which controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element thatfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements are splicing signals, secretion signalsequences, polyadenylation signals, termination signals, RNA exportelements, internal ribosome entry sites, etc. (defined infra).

Transcriptional control signals in eukaryotes comprise “promoter” and“enhancer” elements. Promoters and enhancers consist of short arrays ofDNA sequences that interact specifically with cellular proteins involvedin transcription (Maniatis et al., Science 236:1237 [1987]). Promoterand enhancer elements have been isolated from a variety of eukaryoticsources including genes in yeast, insect and mammalian cells, andviruses (analogous control elements, i.e., promoters, are also found inprokaryotes). The selection of a particular promoter and enhancerdepends on what cell type is to be used to express the protein ofinterest. Some eukaryotic promoters and enhancers have a broad hostrange while others are functional in a limited subset of cell types (forreview see, Voss et al., Trends Biochem. Sci., 11:287 [1986]; andManiatis et al., supra). For example, the SV40 early gene enhancer isvery active in a wide variety of cell types from many mammalian speciesand has been widely used for the expression of proteins in mammaliancells (Dijkema et al., EMBO J. 4:761 [1985]). Two other examples ofpromoter/enhancer elements active in a broad range of mammalian celltypes are those from the human elongation factor 1α gene (Uetsuki etal., J. Biol. Chem., 264:5791 [1989]; Kim et al., Gene 91:217 [1990];and Mizushima and Nagata, Nuc. Acids. Res., 18:5322 [1990]) and the longterminal repeats of the Rous sarcoma virus (Gorman et al., Proc. Natl.Acad. Sci. USA 79:6777 [1982]) and the human cytomegalovirus (Boshart etal., Cell 41:521 [1985]).

As used herein, the term “promoter/enhancer” denotes a segment of DNAwhich contains sequences capable of providing both promoter and enhancerfunctions (i.e., the functions provided by a promoter element and anenhancer element, see above for a discussion of these functions). Forexample, the long terminal repeats of retroviruses contain both promoterand enhancer functions. The enhancer/promoter may be “endogenous” or“exogenous” or “heterologous.” An “endogenous” enhancer/promoter is onewhich is naturally linked with a given gene in the genome. An“exogenous” or “heterologous” enhancer/promoter is one which is placedin juxtaposition to a gene by means of genetic manipulation (i.e.,molecular biological techniques such as cloning and recombination) suchthat transcription of that gene is directed by the linkedenhancer/promoter.

As used herein, the term “secretion signal” refers to any DNA sequencethat when operably linked to a recombinant DNA sequence encodes a signalpeptide which is capable of causing the secretion of the recombinantpolypeptide. In general, the signal peptides comprise a series of about15 to 30 hydrophobic amino acid residues (See, e.g., Zwizinski et al.,J. Biol. Chem. 255(16): 7973-77 [1980], Gray et al., Gene 39(2): 247-54[1985], and Martin et al., Science 205: 602-607 [1979]). Such secretionsignal sequences are preferably derived from genes encoding polypeptidessecreted from the cell type targeted for tissue-specific expression(e.g., secreted milk proteins such as alpha-lactalbumin, alpha-, beta-,and kappa-casein, and whey acidic protein for expression in andsecretion from mammary secretory cells). Secretory DNA sequences,however, are not limited to such sequences. Secretory DNA sequences fromproteins secreted from many cell types and organisms may also be used(e.g., the secretion signals for t-PA, serum albumin, lactoferrin, andgrowth hormone, and secretion signals from microbial genes encodingsecreted polypeptides such as from yeast, filamentous fungi, andbacteria).

Regulatory elements may be tissue specific or cell specific. The term“tissue specific” as it applies to a regulatory element refers to aregulatory element that is capable of directing greater levels ofexpression of a nucleotide sequence of interest in a specific type oftissue (e.g., liver) relative to the level of expression of the samenucleotide sequence of interest in a different type of tissue (e.g.,lung).

Tissue specificity of a regulatory element may be evaluated by, forexample, operably linking a reporter gene to a promoter sequence (whichis not tissue-specific) and to the regulatory element to generate areporter construct, introducing the reporter construct into the genomeof an animal such that the reporter construct is integrated into everytissue of the resulting transgenic animal, and detecting the expressionof the reporter gene (e.g., detecting mRNA, protein, or the activity ofa protein encoded by the reporter gene) in different tissues of thetransgenic animal. The detection of a greater level of expression of thereporter gene in one or more tissues relative to the level of expressionof the reporter gene in other tissues shows that the regulatory elementis “specific” for the tissues in which greater levels of expression aredetected. Thus, the term “tissue-specific” (e.g., liver-specific) asused herein is a relative term that does not require absolutespecificity of expression. In other words, the term “tissue-specific”does not require that one tissue have extremely high levels ofexpression and another tissue have no expression. It is sufficient thatexpression is greater in one tissue than another. By contrast, “strict”or “absolute” tissue-specific expression is meant to indicate expressionin a single tissue type (e.g., liver) with no detectable expression inother tissues.

The term “cell type specific” as applied to a regulatory element refersto a regulatory element which is capable of directing a greater level ofexpression of a nucleotide sequence of interest in a specific type ofcell relative to the level of expression of the same nucleotide sequenceof interest in a different type of cell within the same tissue. The term“cell type specific” when applied to a regulatory element also means aregulatory element capable of promoting selective expression of anucleotide sequence of interest in a region within a single tissue.

Cell type specificity of a regulatory element may be assessed usingmethods well known in the art (e.g., immunohistochemical staining and/orNorthern blot analysis). Briefly, for immunohistochemical staining,tissue sections are embedded in paraffin, and paraffin sections arereacted with a primary antibody specific for the polypeptide productencoded by the nucleotide sequence of interest whose expression isregulated by the regulatory element. A labeled (e.g., peroxidaseconjugated) secondary antibody specific for the primary antibody isallowed to bind to the sectioned tissue and specific binding detected(e.g., with avidin/biotin) by microscopy. Briefly, for Northern blotanalysis, RNA is isolated from cells and electrophoresed on agarose gelsto fractionate the RNA according to size followed by transfer of the RNAfrom the gel to a solid support (e.g., nitrocellulose or a nylonmembrane). The immobilized RNA is then probed with a labeledoligo-deoxyribonucleotide probe or DNA probe to detect RNA speciescomplementary to the probe used. Northern blots are a standard tool ofmolecular biologists.

The term “promoter,” “promoter element,” or “promoter sequence” as usedherein, refers to a DNA sequence which when ligated to a nucleotidesequence of interest is capable of controlling the transcription of thenucleotide sequence of interest into mRNA. A promoter is typically,though not necessarily, located 5′ (i.e., upstream) of a nucleotidesequence of interest whose transcription into mRNA it controls, andprovides a site for specific binding by RNA polymerase and othertranscription factors for initiation of transcription.

Promoters may be constitutive or regulatable. The term “constitutive”when made in reference to a promoter means that the promoter is capableof directing transcription of an operably linked nucleic acid sequencein the absence of a stimulus (e.g., heat shock, chemicals, etc.). Incontrast, a “regulatable” promoter is one which is capable of directinga level of transcription of an operably linked nucleic acid sequence inthe presence of a stimulus (e.g., heat shock, chemicals, etc.) which isdifferent from the level of transcription of the operably linked nucleicacid sequence in the absence of the stimulus.

The presence of “splicing signals” on an expression vector often resultsin higher levels of expression of the recombinant transcript. Splicingsignals mediate the removal of introns from the primary RNA transcriptand consist of a splice donor and acceptor site (Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, New York [1989], pp. 16.7-16.8). A commonly usedsplice donor and acceptor site is the splice junction from the 16S RNAof SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cellsrequires expression of signals directing the efficient termination andpolyadenylation of the resulting transcript. Transcription terminationsignals are generally found downstream of the polyadenylation signal andare a few hundred nucleotides in length. The term “poly A site” or “polyA sequence” as used herein denotes a DNA sequence that directs both thetermination and polyadenylation of the nascent RNA transcript. Efficientpolyadenylation of the recombinant transcript is desirable astranscripts lacking a poly A tail are unstable and are rapidly degraded.The poly A signal utilized in an expression vector may be “heterologous”or “endogenous.” An endogenous poly A signal is one that is foundnaturally at the 3′ end of the coding region of a given gene in thegenome. A heterologous poly A signal is one that is isolated from onegene and placed 3′ of another gene. A commonly used heterologous poly Asignal is the SV40 poly A signal. The SV40 poly A signal is contained ona 237 bp BamHI/BclI restriction fragment and directs both terminationand polyadenylation (Sambrook, supra, at 16.6-16.7).

Eukaryotic expression vectors may also contain “viral replicons ” or“viral origins of replication.” Viral replicons are viral DNA sequencesthat allow for the extrachromosomal replication of a vector in a hostcell expressing the appropriate replication factors. Vectors thatcontain either the SV40 or polyoma virus origin of replication replicateto high “copy number” (up to 10⁴ copies/cell) in cells that express theappropriate viral T antigen. Vectors that contain the replicons frombovine papillomavirus or Epstein-Barr virus replicate extrachromosomallyat “low copy number” (˜100 copies/cell). However, it is not intendedthat expression vectors be limited to any particular viral origin ofreplication.

As used herein, the term “long terminal repeat” of “LTR” refers totranscriptional control elements located in or isolated from the U3region 5′ and 3′ of a retroviral genome. As is known in the art, longterminal repeats may be used as control elements in retroviral vectors,or isolated from the retroviral genome and used to control expressionfrom other types of vectors.

As used herein, the term “secretion signal” refers to any DNA sequencewhich when operably linked to a recombinant DNA sequence encodes asignal peptide which is capable of causing the secretion of therecombinant polypeptide. In general, the signal peptides comprise aseries of about 15 to 30 hydrophobic amino acid residues (See, e.g.,Zwizinski et al., J. Biol. Chem. 255(16): 7973-77 [1980], Gray et al.,Gene 39(2): 247-54 [1985], and Martial et al., Science 205: 602-607[1979]). Such secretion signal sequences are preferably derived fromgenes encoding polypeptides secreted from the cell type targeted fortissue-specific expression (e.g., secreted milk proteins for expressionin and secretion from mammary secretory cells). Secretory DNA sequences,however, are not limited to such sequences. Secretory DNA sequences fromproteins secreted from many cell types and organisms may also be used(e.g., the secretion signals for t-PA, serum albumin, lactoferrin, andgrowth hormone, and secretion signals from microbial genes encodingsecreted polypeptides such as from yeast, filamentous fungi, andbacteria).

As used herein, the terms “RNA export element” or “Pre-mRNA ProcessingEnhancer (PPE)” refer to 3′ and 5′ cis-acting post-transcriptionalregulatory elements that enhance export of RNA from the nucleus. “PPE”elements include, but are not limited to Mertz sequences (described inU.S. Pat. Nos. 5,914,267 and 5,686,120, all of which are incorporatedherein by reference) and woodchuck mRNA processing enhancer (WPRE;WO99/14310, incorporated herein by reference).

As used herein, the term “polycistronic” refers to an mRNA encoding morethan polypeptide chain (See, e.g., WO 93/03143, WO 88/05486, andEuropean Pat. No. 117058, all of which is incorporated herein byreference). Likewise, the term “arranged in polycistronic sequence”refers to the arrangement of genes encoding two different polypeptidechains in a single mRNA.

As used herein, the term “internal ribosome entry site” or “IRES” refersto a sequence located between polycistronic genes that permits theproduction of the expression product originating from the second gene byinternal initiation of the translation of the dicistronic mRNA. Examplesof internal ribosome entry sites include, but are not limited to, thosederived from foot and mouth disease virus (FDV), encephalomyocarditisvirus, poliovirus and RDV (Scheper et al., Biochem. 76: 801-809 [1994];Meyer et al., J. Virol. 69: 2819-2824 [1995]; Jang et al., 1988, J.Virol. 62: 2636-2643 [1998]; Haller et al., J. Virol. 66: 5075-5086[1995]). Vectors incorporating IRES's may be assembled as is known inthe art. For example, a retroviral vector containing a polycistronicsequence may contain the following elements in operable association:nucleotide polylinker, gene of interest, an internal ribosome entry siteand a mammalian selectable marker or another gene of interest. Thepolycistronic cassette is situated within the retroviral vector betweenthe 5′ LTR and the 3′ LTR at a position such that transcription from the5′ LTR promoter transcribes the polycistronic message cassette. Thetranscription of the polycistronic message cassette may also be drivenby an internal promoter (e.g., cytomegalovirus promoter) or an induciblepromoter, which may be preferable depending on the use. Thepolycistronic message cassette can further comprise a cDNA or genomicDNA (gDNA) sequence operatively associated within the polylinker. Anymammalian selectable marker can be utilized as the polycistronic messagecassette mammalian selectable marker. Such mammalian selectable markersare well known to those of skill in the art and can include, but are notlimited to, kanamycin/G418, hygromycin B or mycophenolic acid resistancemarkers.

As used herein, the term “retrovirus” refers to a retroviral particlewhich is capable of entering a cell (i.e., the particle contains amembrane-associated protein such as an envelope protein or a viral Gglycoprotein which can bind to the host cell surface and facilitateentry of the viral particle into the cytoplasm of the host cell) andintegrating the retroviral genome (as a double-stranded provirus) intothe genome of the host cell.

As used herein, the term “retroviral vector” refers to a retrovirus thathas been modified to express a gene of interest. Retroviral vectors canbe used to transfer genes efficiently into host cells by exploiting theviral infectious process. Foreign or heterologous genes cloned (i.e.,inserted using molecular biological techniques) into the retroviralgenome can be delivered efficiently to host cells which are susceptibleto infection by the retrovirus. Through well known geneticmanipulations, the replicative capacity of the retroviral genome can bedestroyed. The resulting replication-defective vectors can be used tointroduce new genetic material to a cell but they are unable toreplicate. A helper virus or packaging cell line can be used to permitvector particle assembly and egress from the cell. Such retroviralvectors comprise a replication-deficient retroviral genome containing anucleic acid sequence encoding at least one gene of interest (i.e., apolycistronic nucleic acid sequence can encode more than one gene ofinterest), a 5′ retroviral long terminal repeat (5′ LTR); and a 3′retroviral long terminal repeat (3′ LTR).

The term “pseudotyped retroviral vector” refers to a retroviral vectorcontaining a heterologous membrane protein. The term“membrane-associated protein” refers to a protein (e.g., a viralenvelope glycoprotein or the G proteins of viruses in the Rhabdoviridaefamily such as VSV, Piry, Chandipura and Mokola) which are associatedwith the membrane surrounding a viral particle; thesemembrane-associated proteins mediate the entry of the viral particleinto the host cell. The membrane associated protein may bind to specificcell surface protein receptors, as is the case for retroviral envelopeproteins or the membrane-associated protein may interact with aphospholipid component of the plasma membrane of the host cell, as isthe case for the G proteins derived from members of the Rhabdoviridaefamily.

The term “heterologous membrane-associated protein” refers to amembrane-associated protein which is derived from a virus which is not amember of the same viral class or family as that from which thenucleocapsid protein of the vector particle is derived. “Viral class orfamily” refers to the taxonomic rank of class or family, as assigned bythe International Committee on Taxonomy of Viruses.

The term “Rhabdoviridae” refers to a family of enveloped RNA virusesthat infect animals, including humans, and plants. The Rhabdoviridaefamily encompasses the genus Vesiculovirus which includes vesicularstomatitis virus (VSV), Cocal virus, Piry virus, Chandipura virus, andSpring viremia of carp virus (sequences encoding the Spring viremia ofcarp virus are available under GenBank accession number U18101). The Gproteins of viruses in the Vesiculovirus genera are virally-encodedintegral membrane proteins that form externally projecting homotrimericspike glycoproteins complexes that are required for receptor binding andmembrane fusion. The G proteins of viruses in the Vesiculovirus generahave a covalently bound palmititic acid (C₁₆) moiety. The amino acidsequences of the G proteins from the Vesiculoviruses are fairly wellconserved. For example, the Piry virus G protein share about 38%identity and about 55% similarity with the VSV G proteins (severalstrains of VSV are known, e.g., Indiana, New Jersey, Orsay, San Juan,etc., and their G proteins are highly homologous). The Chandipura virusG protein and the VSV G proteins share about 37% identity and 52%similarity. Given the high degree of conservation (amino acid sequence)and the related functional characteristics (e.g., binding of the virusto the host cell and fusion of membranes, including syncytia formation)of the G proteins of the Vesiculoviruses, the G proteins from non-VSVVesiculoviruses may be used in place of the VSV G protein for thepseudotyping of viral particles. The G proteins of the Lyssa viruses(another genera within the Rhabdoviridae family) also share a fairdegree of conservation with the VSV G proteins and function in a similarmanner (e.g., mediate fusion of membranes) and therefore may be used inplace of the VSV G protein for the pseudotyping of viral particles. TheLyssa viruses include the Mokola virus and the Rabies viruses (severalstrains of Rabies virus are known and their G proteins have been clonedand sequenced). The Mokola virus G protein shares stretches of homology(particularly over the extracellular and transmembrane domains) with theVSV G proteins which show about 31% identity and 48% similarity with theVSV G proteins. Preferred G proteins share at least 25% identity,preferably at least 30% identity and most preferably at least 35%identity with the VSV G proteins. The VSV G protein from which NewJersey strain (the sequence of this G protein is provided in GenBankaccession numbers M27165 and M21557) is employed as the reference VSV Gprotein.

As used herein the term, the term “in vitro” refers to an artificialenvironment and to processes or reactions that occur within anartificial environment. In vitro environments can consist of, but arenot limited to, test tubes and cell cultures. The term “in vivo” refersto the natural environment (e.g., an animal or a cell) and to processesor reaction that occur within a natural environment.

As used herein, the term “purified” refers to molecules, either nucleicor amino acid sequences, that are removed from their naturalenvironment, isolated or separated. An “isolated nucleic acid sequence”is therefore a purified nucleic acid sequence. “Substantially purified”molecules are at least 60% free, preferably at least 75% free, and morepreferably at least 90% free from other components with which they arenaturally associated.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to transgenic animals that expressexogenous growth factors in their milk, and in particular to pigs thatexpress exogenous growth factors in their milk. In some embodiments, thegrowth factor is selected from the group including, but not limited to,IGF-I (insulin-like growth factor I), IGF-II (insulin like growth factorII), EGF (epidermal growth factor), PDGF (platelet derived growthfactor), FGF (fibroblast growth factor), and TGF (transforming growthfactor). In some embodiments, the gene is human IGF-I (e.g., SEQ IDNO:2). In other embodiments, the nucleic acid encoding the IGF-1 genehybridizes to SEQ ID NO:2 under conditions of low to high stringency. Inpreferred embodiments, the growth factor gene is part of a geneconstruct (e.g., SEQ ID NO:1). In some preferred embodiments, the geneconstruct further comprises a mammary-specific promoter/enhancer region(e.g., including but not limited to those derived from the casein, wheyacidic, lactoferrin, and lactoglobin promoters and variants and homologsthereof).

In some embodiments, the present invention provides transgenic pigs thatproduce a growth factor (e.g., IGF-I) in their milk. In preferredembodiments, piglets suckling milk from transgenic pigs have anincreased rate of growth compared to piglets suckling fromnon-transgenic pigs. Additionally, in preferred embodiments, pigletssuckling from transgenic pigs have increased levels of intestinallactase and improved intestinal health.

I. Growth Factors

A. Growth Factor Genes

In some embodiments, the present invention provides transgenic animals(e.g., pigs) that expresses exogenous growth factors (e.g., IGF-I,IGF-II, and EGF and homologs thereof) in their milk. IGF-I is highlyconserved, with the human, porcine, ovine and bovine sequences showing100% homology at the amino acid level (Tavakkol et al., MolecEndocrinol., 2:674 [1988]). IGF-I and IGF-II are approximately 7.5 kDapeptides which exhibit 70% amino acid homology with each other and 50%homology with proinsulin.

IGF-I and IGF-II are products of two distinct genes. IGF-I and IGF-IIinteract with specific receptors, the type I and type II IGF receptors,respectively, but also cross react to some degree with each other'sreceptors. Both interact with binding proteins, but there aredifferences in the affinity of different binding proteins for IGF-I vs.IGF-II. IGF-I and IGF-II participate in many of the same physiologicalactions, but have discrete actions of their own as well.

In some embodiments, the methods and compositions of the presentinvention utilize the human IGF-I gene (e.g., SEQ ID NO:2). In otherembodiments, the methods and compositions of the present inventionutilize another growth factor (e.g., a different IGF-I gene, an IGF-IIgene, or an EGF gene), including but not limited to those described inTable 1 below.

TABLE 1 Growth Factor Genes Organism Gene Accession Number(s) HumanIGF-I E01712; E01709; E01168; E00602; E00306; E00305; E00304; E00303;S85346; X03422; X03421; X03420; X03419; X57025; X03563; U40870; M59812;M37484; M37483; M27544; AH002705; M14156; M14155; M14154; M14153;M12659; Pig IGF-I X64400 Mouse IGF-I AF056187; X04480; M28139 Rat IGF-ID00698; AA926152; S68686; S68264; S43941; X06108; X06107; M15481;M17335; M15480; M27293; AH002176 Japanese Quail IGF-I AF260131; S75247Goat IGF-I SEG_GOTIGFI; D26118; D26116; D26119; D26117; D11378 OstrichIGF-I AB035804 Japanese Flounder IGF-I AJ010602; AJ010603 Musk ShrewIGF-I D43957 Goldfish IGF-I AF001006; AF001005 Black Seabream IGF-IAF030573 Scorpion fish IGF-I Y12583 Turkey IGF-I AF074980 chum SalmonIGF-I AF063216; AH004580; S70778; S70777 Mozambique Tilapia IGF-IAH006116; AF033800; AF033799; AF033798; AF33797; AF033796 Rabbit IGF-IU75390 Sheep IGF-I X51403; X51358; X51357; M31734; M30653; M31735;M31736 Catfish IGF-I X79077; X79244; Guinea Pig IGF-I X52951 XenopusIGF-I M29857 Chinook Salmon IGF-I U15962; U15961; U15960; U14536 CohoSalmon IGF-I M32792 Chicken IGF-I M32791 Bovine IGF-II E01192; X53867Sheep IGF-II Y16533; U00668; U00667; U00666; U00665; U00664; U00663;M89789; M89788 Human IGF-II X03427; X07868; S77035; X03426; X03425;X03424; X03562; X05330; X05331; X07867; X03423; A18005; A18004;AH002704; M22373; M22372; AH002703; M14118; M14117; M14116; M13970;M17862 Mouse IGF-II BE335824; AW822068; AW743221; AW742693; AW742516;AW742405; NM_010514; AI562175; AI325269; A1157816; U71085; AI120019;AA409837; AA407349; C80191; AA958906; AA959114; X71922; X71921; X71920;X71919; X71918; M36334; M36333; AH001929; M36332; M36331; M36330; M36329Mozambique Tilapia IGF-II AH006117; AF033804; AF033803; AF033802;AF033801 Zebra Finch IGF-II AJ223165 Rat IGF-II AH005814; M13871;M13870; M13869; M13868; M22474; AH002187; M29880; M29879; M29878; M17960Chum Salmon IGF-II X9725 Barramundi Perch IGF-II AF007943 Chicken IGF-IIAH005039; S82962 Bovine EGF L12259 Pig EGF AF079769; AF079768 Mouse EGFNM019397; NM_010113; X74038; J00380 Rat EGF NM_012842 Human EGFNM_005928; NM_001963; E02238; E01114; E00779; E00309; E00208; L17030;L17029 Sheep EGF X89506 Chicken EGF Y09264 Human PDGF AF244813;NM_002608; AF169595; AH007345; S50869; 261626; S51624; E02395; X02811;X03795; X06374 Mouse PDGF NM_008808; AF286725; AH004413 Chicken FGFAB030229; U55189 Bovine FGF AJ003123; M13440; M13439 Sheep FGF AF213396Human FGF NM_019851; AB021975; AB030648; AB044277; NM_019113 Rat FGFNM_019199; NM_019198 Mouse FGF AB025718; AB029498; NM_010202; NM_010201Bovine TGF M36271 Porcine TGF X14150; X12373; X70142 Mouse TGFNM_019678; NM_009368 Human TGF NM_003239; X05844; NM_009368

B. Biological Activity of Growth Factors

In some embodiments, the present invention provides animals (e.g., pigs)that express a growth factor gene in their milk during lactation. Insome embodiments, the transgenic animal is a female. In otherembodiments, the transgenic animal is a lactating male animal. Methodsare known in the art for inducing lactation in animals. For example,lactation can be induced in bovines by repeated injections of estradioland progesterone (See e.g., Smith and Schanbacher, J. Dairy Sci., 56:758[1973]; and Sawyer et al., J. Dairy Sci., 69:1536 [1986]). It iscontemplated that offspring (e.g., piglets) that suckle transgenicanimals expressing growth factors in their milk will have improvedintestinal health and development.

1. Effect of Growth Factors on Mammary Growth and Nutrient Uptake

The present invention is not limited to any one mechanism. Indeed, anunderstanding of the mechanism is not necessary to practice the presentinvention. Nonetheless, IGF-I is thought to mediate a portion of theeffects of growth hormone on lactation (Cohick, J. Dairy Sci., 81:1769[1998]). It is contemplated that IGF-I has an effect in regulatingmammary growth and differentiation. IGF-I has been shown to stimulatecellular growth and DNA synthesis in cultured bovine and ovine mammarytissues (Shamay et al., Endocrinology, 123:804 [1988]; Wunder et al., J.Endocrinology, 123:319 [1989]). IGF-I has also been shown to havemitogenic activity and to be an inhibitor of mammary apoptosis (Cohick,J. Dairy Sci., 81:1769 [1998]; Neuenschwander et al., J. Clin Invest.,97:2225 [1996]; Rosfjord and Dickson, J. Mamm. Gland Biol. Neoplasia,4:229 [1999]).

Mammary and intestinal tissue share several key nutrient uptake systems.Several of these transporters utilize an electrochemical gradient of Na⁺(Shennan, J. Mam. Gland Biol. Neoplasia, 3:247 [1998]). For example,glucose uptake by the mammary gland occurs by both the GLUT and SGLTsystems. Glucose uptake across the GLUT transporter occurs byfacilitative diffusion, whereas the SGLT transporter utilizes theNA+Gradient (Shennan, J. Mam. Gland Biol. Neoplasia, 3:247 [1998]).Sodium-dependent glucose uptake in pig intestine is increased by IGF-I(Alexander and Carey, Am. J. Physiol., 277:G619 [1999]). The presentinvention is not limited to any one mechanism. Indeed, an understandingof the mechanism is not necessary to practice the present invention.Nonetheless, it is contemplated that expression of a growth factor(e.g., IGF-I) in the mammary gland of an animal (e.g., a pig) modulatesnutrient transport within mammary epithelial tissue. Specifically, it iscontemplated that the overexpression of a growth factor (e.g., IGF-I) inthe mammary gland will enhance early mammary development and mammaryfunction in lactation, and will inhibit involution.

Example 5 describes experiments investigating the effect of one growthfactor (IGF-I) on mammary gland development and nutrient uptake. It iscontemplated that the mammary glands of sows overexpressing IGF-I willshow enhanced proliferation and reduced apoptosis compared to controlnon-transgenic siblings. For example, it is contemplated that theoverexpression of IGF-I in the mammary glands of sows will decreaseinvolution of the mammary glands, resulting in longer lactation andincreased milk production. Additionally, it is contemplated that thesealterations in cellular proliferation and cell death will lead to anincrease in mammary cellularity and changes in mammary morphology.Furthermore, it is contemplated that Na⁺ coupled nutrient transport aswell as kinetic properties of valine and glucose uptake will be enhancedin tissue from transgenic, but not control sows.

2. Effect of Exogenous Growth Factors on Sow Milk

In some embodiments, the present invention provides methods ofincreasing piglet growth and intestinal lactase activity. Example 3describes the measurement of several properties of milk from transgenicsows overexpressing IGF-I compared to non-transgenic control sows.Properties investigated include total milk production, IGF-I and IGF-Ibinding protein levels in milk, total milk solids, milk protein content,and milk fat content.

Milk production was found to be higher in transgenic sows relative tocontrol sows on day 3 of lactation (Example 3A; FIG. 4). The IGF-Icontent of colostrum of transgenic sows was found to approximately 10fold higher than non-transgenic control sows (Example 3B; FIG. 5). Theincreased IGF-I content was maintained throughout lactation. The milk oftransgenic sows was found to contain increased levels of IGFBP5 relativeto non-transgenic control animals (Example 3C). Protein levels werehigher in the milk of transgenic sows on days 0, and 1 of lactation andwere higher in control samples on days 8 and 24 of lactation (Example3E; FIG. 7). Milk from transgenic sows had a higher fat content at days0-2 of lactation (Example 3G; FIG. 9). The level of milk solids waslower in transgenic sows than control sows at day 0 of lactation(Example 3D; FIG. 6). Levels of milk solids were similar in transgenicand control samples for the remainder of the study. The lactose contentof milk samples from transgenic and non-transgenic sows was not found tobe significantly different (Example 3F; FIG. 8).

3. Effect of Growth Factors on Neonatal Intestinal Development

The present invention is not limited to any one mechanism. Indeed, anunderstanding of the mechanism is not necessary to practice the presentinvention. Nonetheless, it is contemplated that the expression of agrowth factor (e.g., IGF-I) in the milk of transgenic sows will have abeneficial effect on intestinal development and health of sucklingpiglets. Specifically, it is contemplated that piglets suckling fromsows expressing a growth factor (e.g., IGF-I) will have increasedresistance to intestinal disease, increased nutrient metabolism, andincreased hardiness during weaning.

Oral administration of IGF-I was shown to increase intestinal lactaseactivity (Example 1; FIGS. 1 and 2). Piglets suckling transgenic sowsexpressing IGF-I in their milk were found to have increased intestinallactase activity (Example 4B; FIG. 11).

Example 6 describes experiments designed to further investigate theeffect of IGF-I on piglet intestinal morphology, disaccharidase geneexpression, nutrient transport and ion secretion, nutrient transportergene expression, and nutrient transporter protein abundance. It iscontemplated that intestinal villus morphology and disaccharidaseactivity will be greater in piglets suckling transgenic sows. It isfurther contemplated that Na⁺-coupled glucose and glutamine transportwill be greater in the intestines of piglets suckling transgenic sows.In addition, it is contemplated that the maximum uptake rate of pigletssuckling transgenic sows will be greater because of the enhanced effluxof intracellular Na⁺. Additionally, it is contemplated that pigletssuckling transgenic sows will have increased cell division in the smallintestine. Finally, it is contemplated that increased Na⁺,K⁺-ATPaseactivity and protein levels will be observed.

It is contemplated that piglets suckling from transgenic sows expressinga growth factor (e.g., IGF-I) in their milk will have increasedintestinal health and resistance to intestinal diseases. Specifically,it is contemplated that piglets will have increased resistance toscours. Scours is a common pathogenic intestinal disease of sucklingpiglets. Scouring may be caused by pathogens including, but not limitedto rotovirus, coronavirus, E. coli, and salmonella.

II. Methods of Generating Transgenic Animals

A. Gene Constructs

In some embodiments, transgenic animals (e.g., pigs) are generated usingthe gene construct described in SEQ ID NO:1. In some embodiments, thegene construct contains the human IGF-I gene (SEQ ID NO:2). In otherembodiments, the gene construct contains a growth factor gene,including, but not limited to those described in Table 1.

In some embodiments, the gene construct further comprises a promoter. Inpreferred embodiments, the promoter comprises a mammary preferential orspecific promoter. In some embodiments, the mammary preferential orspecific promoter is selected from the group including, but not limitedto alpha-lactalbumin, casein promoters (e.g., alpha S1, beta-, andkappa-), whey acidic promoter, and lactoferrin promoter (described inU.S. Pat. Nos. 6,004,805; 6,027,722; 5,304,489; 5,565,362; 5,831,141;4,873,316; and European Patent No. 264,166; all of which are hereinincorporated by reference). In some embodiments, the promoter comprisesthe bovine alpha-lactalbumin promoter. Some mammary specific promoters(e.g., lactoferrin) direct expression only lactation (See e.g., Goodmanand Schanbacher, Biochem Biophys Res Commun., 180:75 [1991]).

In some embodiments of the present invention, the gene construct furthercomprises a signal peptide to signal secretion of the protein ofinterest. The sequences of several suitable signal peptides are known inthe art, including, but not limited to, those derived from tissueplasminogen activator, human growth hormone, lactoferrin, alphaS1-casein, and alpha-lactalbumin.

B. Methods of Introducing DNA into the Genome of Animals

In some embodiments, the present invention provides a transgenic animal(e.g., a pig) expressing a growth factor (e.g., IGF-I) in their milk.The present invention is not limited to any one method of generatingtransgenic animals. In some embodiments, the transgenic animals aregenerated by pronuclear microinjection. In other embodiments, thetransgenic animals are generated by nuclear transfer. In still furtherembodiments, the transgenic animals are generated by retroviralinfection of oocytes or embryos.

1. Pronuclear Microinjection

In some embodiments, the transgenic animals of the present invention aregenerated by pronuclear microinjection (Bleck et al., J. Anim. Sci.,76:3072 [1998]; also described in U.S. Pat. Nos. 6,066,725, 5,523,226;5,453,457; 4,873,191; 4,736,866, all of which are herein incorporated byreference). In pronuclear microinjection, several hundred copies of DNAare injected into the male pronucleus of the zygote. DNA integrationoccurs during replication as a repair function of the host DNA.

In some embodiments, pronuclear microinjection is performed on thezygote 12 hours post fertilization. Uptake of such genes may be delayedfor several cell cycles. The consequence of this is that depending onthe cell cycle of uptake, only some cell lineages may carry thetransgene, resulting in mosaic offspring. If desired, mosaic animals canbe bred to form true germline transgenic animals.

In one illustrative example of the present invention (Example 2),transgenic pigs were generated by pronuclear injection of the geneconstruct described in SEQ ID NO:1. In this example, 400 embryos wereinjected with the gene construct. Of 71 live births (from 14pregnancies), 4 transgenic animals were identified, for a 5.6%efficiency of transgenic animals generated.

2. Nuclear Transfer

Nuclear transfer, dubbed “cloning” (described in U.S. Pat. Nos.6,011,197; 5,496,720, 4,994,384; and 5,057,420, and WO 97/07669, WO97/07668, and WO 95/17500; all of which are herein incorporated byreference), utilizes a nucleus extracted from non-germline cells toreplace the nucleus of an oocyte from a donor animal. Nuclear transfercan be used as a means of gene introduction. In this case, a cell lineis used as the starting point for gene introduction and then nuclei fromthis line are transferred into embryos for propagation. Genes can beintroduced to the cell line using any suitable method (e.g.,electroporation, transfection or injection)

3. Retroviral Infection

In some embodiments, the transgenic animals of the present invention aregenerated by retroviral gene transfer (Chan et al., PNAS, 95:14028[1998]) and U.S. Pat. No. 6,080,912; herein incorporated by reference).In this method, an exogenous nucleic acid (e.g., IGF-I) is introducedinto pre-maturation oocytes, mature unfertilized oocytes, or zygotesusing retroviral vectors. In preferred embodiments, unfertilized oocytesare infected, permitting the integration of the recombinant provirusprior to the division of the one cell embryo. Thus, all cells in theembryo will contain the proviral sequences.

Retroviruses are enveloped (i.e., surrounded by a host cell-derivedlipid bilayer membrane) single-stranded RNA viruses that infect animalcells. When a retrovirus infects a cell, its RNA genome is convertedinto a double-stranded linear DNA form (i.e., it is reversetranscribed). The DNA form of the virus is then integrated into the hostcell genome as a provirus.

In preferred embodiments, pseudotyped retroviral vectors which containthe G protein of VSV as the membrane associated protein are used for theproduction of transgenic animals. Unlike retroviral envelope proteinswhich bind to a specific cell surface protein receptor to gain entryinto a cell, the VSV G protein interacts with a phospholipid componentof the plasma membrane (Mastromarino et al., J. Gen. Virol. 68:2359[1987]). Because entry of VSV into a cell is not dependent upon thepresence of specific protein receptors, VSV has an extremely broad hostrange. Pseudotyped retroviral vectors bearing the VSV G protein have analtered host range characteristic of VSV (i.e., they can infect almostall species of vertebrate, invertebrate and insect cells). Importantly,VSV G-pseudotyped retroviral vectors can be concentrated 2000-fold ormore by ultracentrifugation without significant loss of infectivity(Burns et al., Proc. Natl. Acad. Sci. USA 90:8033 [1993]).

In some embodiments, the retroviral vectors are packaged andmicroinjected into the perivitelline space of oocytes (e.g., in vitro orin vivo matured oocytes). The oocytes are then fertilized. In otherembodiments, zygotes are injected with the packaged retroviral vectors.The resulting embryos or zygotes can then be transferred into recipientanimals.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: M (molar); mM (millimolar); μM (micromolar); nM(nanomolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); gm (grams); mg (milligrams); μg (micrograms); pg(picograms); L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C(degrees Centigrade); AMP (adenosine 5′-monophosphate); BSA (bovineserum albumin); cDNA (copy or complimentary DNA); CS (calf serum); DNA(deoxyribonucleic acid); ssDNA (single stranded DNA); dsDNA (doublestranded DNA); dNTP (deoxyribonucleotide triphosphate); KB (kilobase);bp (base pair); LH (luteinizing hormone); NIH (National Institutes ofHealth, Besthesda, Md.); RNA (ribonucleic acid); PBS (phosphate bufferedsaline); g (gravity); OD (optical density); HEPES(N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]); HBS (HEPESbuffered saline); PBS (phosphate buffered saline); SDS (sodiumdodecylsulfate); Tris-HCl(tris[Hydroxymethyl]aminomethane-hydrochloride); Klenow (DNA polymeraseI large (Klenow) fragment); EGTA (ethylene glycol-bis(β-aminoethylether) N,N,N′,N′-tetraacetic acid); EDTA (ethylenediaminetetraceticacid); bla (β-lactamase or ampicillin-resistance gene); lacI (lacrepressor); X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside); ATCC(American Type Culture Collection, Rockville, Md.); GIBCO/BRL(GIBCO/BRL, Grand Island, N.Y.); Perkin-Elmer (Perkin-Elmer, Norwalk,Conn.); and Sigma (Sigma Chemical Company, St. Louis, Mo.); Intervet(Intervet, Millsboro, Del.); Packard (Packard Instruments, Meriden,Conn.); Micron Separation (Micron Separation, Inc., Westborough, Mass.);Pierce (Pierce, Rockford, Ill.); Novocastra (Novocastra Laboratories,Burlingham, Calif.); Fryer (Fryer Company, Inc., Carpentersville, Ill.);Universal Imaging (Universal Imaging Corp., Westchester, Pa.);Physiologic (Physiologic Instruments, Inc., San Diego, Calif.);Molecular Dynamics (Molecular Dynamics, Sunnyvale, Calif.); OncogeneScience (Oncogene Science Inc., Uniondale, N.Y.); Amersham (Amersham,Arlington Heights, Ill.).

EXAMPLE 1 Effect of Oral IGF-I on Piglet Development

Newborn, colostrum-deprived piglets were fed sow milk replacer alone orsupplemented with 0, 0.25, 0.5, or 1.0 mg/L recombinant human IGF-I for14 days. The effect of the oral IGF-I on lactase activity in pigletintestines was measured. The results are shown in FIGS. 1 and 2.

FIG. 1 shows lactase activity in various segments of the intestine incontrol piglets and piglets fed 1.0 mg/L IGF-I for 14 days. Segment 1represents lactase levels in the duodenum; segments 2-7 represent theproximal jejunum; segments 8-11 represent the distal jejunum; andsegments 12-13 represent the ileum. FIG. 2 shows lactase activity invarious segments of the intestine in response to varied levels of oralIGF-I.

EXAMPLE 2 Generation of IGF-I Transgenic Pigs

A. α-LA/IGF-I Gene Construct

Pronuclear stage porcine embryos were injected with a α-LA/IGF-I geneconstruct (SEQ ID NO:1; FIG. 3). The hybrid construct contains 210 bp ofhuman IGF-I coding region (Exon 4) inserted in exon 1 of the bovineα-LAC gene. The construct also contains 2 kb of 5′ flanking region fromthe bovine α-LAC gene, the bovine α-LAC signal peptide and promoter,Exons 2-4 of the bovine α-LAC gene including the polyA signal, and 329bp of 3′ flanking region.

This gene construct was created by inserting the cDNA encoding matureIGF-I behind the bovine α-lactalbumin signal peptide. Once this fusionwas created it was then inserted into exon 1 of the bovine α-lactalbumingene. The resulting mRNA produced from this gene construct resemblesendogenous α-lactalbumin mRNA, except there is the additional IGF-Icoding region contained within the exon 1 portion of the mRNA. Theexpression of this mRNA is under the control of the mammary specificbovine α-lactalbumin 5′ flanking region.

The protein translated from this mRNA is a fusion of the bovineα-lactalbumin signal peptide and the human IGF-I mature protein. Thesignal peptide allows secretion of mature IGF-I into the milk aftercleavage of the signal peptide in the rough endoplasmic reticulum.

B. Collection of Embryos

Embryos were collected and injected using previously described methods(Bleck et al., J. Anim. Sci., 76:3072 [1998]). Duroc, Yorkshire, andDuroc X Yorkshire gilts were injected with PG 600 (Intervet) at 170 to210 days of age. Gilts that responded to the injection by exhibitingstanding estrus continued in the study. These animal were injected withPMSG (Sigma) 16 days after standing estrus and then injected with hCG 72hours later. Animal exhibiting standing estrus were artificiallyinseminated. Embryos were collected 54 hours after hCG injection bysurgical embryo collection from the oviduct. 908 Embryos were flushedfrom the oviduct using Beltsville embryo culture medium (Dobrinsky etal., Biol. Reprod., 55:1069 [1996]). The embryos were centrifuged at15,000×g for 5-10 minutes to visualize the pronuclei.

C. Injection and Transfer

Four hundred pronuclear embryos were injected with the α-LA/IGF-I geneconstruct. The injected DNA was at a concentration of approximately 4ng/μl in microinjection buffer (10 mM Tris, 0.1 mM EDTA, pH 7.4).Approximately 20 normal-appearing injected embryos were transferred toeach recipient animal. Recipient gilts were animals showing standingestrus within a day of the donor animal. Thirty six sows receivedembryos, resulting in 14 pregnancies and 71 live births.

The piglets were screened for the presence of the transgene using PCR.DNA was extracted from ear and tail and PCR was performed using themethods of Bleck and Bremel (J. Dairy Sci., 77:1897 [1994]). Transgenicpigs were characterized by the presence of a 360 bp product. Four of thepiglets were transgenic, resulting in a 5.6% transgenic efficiency.

EXAMPLE 3 Properties of Milk of IGF-I Transgenic Sows

Transgenic gilts and control gilts were bred to the same non-transgenicboar and allowed to farrow. Litter sizes were adjusted to 10 piglets.Milk samples were obtained by manual expression on days 3, 7, 14, and 21days post-lactation. Piglets were separated from the sow for one hourprior to milking. Sows were injected intravenously with 1.0 IU ofoxytocin to promote milk let-down. Milk (50-100 ml) was collected intosterile containers and frozen immediately. Blood samples were collectedfrom the ear vein for IGF-I and IGFBP studies.

A. Milk production of Transgenic Sows

Milk yield was measured using the weigh-suckle-weigh technique (Lewis etal., J. Animal Sci., 47:634 [1978]). All of the piglets in a litter wereremoved from the sow for 1 hour, weighed, and returned to the sow andallowed to suckle for 15 minutes. Following suckling, the piglets wereseparated from the sow, weighed again, and kept separate from the sowuntil the next suckling period. The procedure was repeated every hourfor 6 hours. The difference in body weight of the piglets before andafter suckling was taken as the level of milk production.

FIG. 4 shows milk production of transgenic and control sows. Milkproduction was higher in transgenic sows relative to control sows on day3 of lactation.

B. IGF-I Content in the Milk of Transgenic Sows

Milk samples were collected from transgenic, control, and reference(Donovan et al., Pediatric Res., 36:159 [1994]) sows between 12 hoursand 20 days postpartum. Milk samples were de-fatted and casein wasremoved by acidification and centrifugation. Milk IGF-I was separatedfrom IGFBP by chromatography in 0.2 mol/L formic acid (Donovan et al.,1994, supra). IGF-I fractions were collected and lyophilized. IGFrecovery from the column was 90%. The concentration of IGF-I wasmeasured using [¹²⁵I]-IGF-I as a competitive ligand and a polyclonalanti-human IGF-I antibody (NIH). After overnight incubation, 1% bovineIgG and 20% PEG (Sigma) was added and the tubes were centrifuged. Boundradioactivity was measured using a gamma counter (Cobra-II Autogammacounter, Packard). Concentrations were determined relative to a standardcurve.

Data are shown in FIG. 5. IGF-I content was approximately 10 fold higherin the colostrum of transgenic sows than non-transgenic control sows.The increased IGF-I content in transgenic sows was maintained atapproximately 60-fold higher in mature mild throughout lactation.

C. IGF-I Binding Protein Levels in Transgenic Sows

Serum and milk IGF binding protein (IGFBP) levels were measured bywestern ligand blotting (Donovan, 1994). Serum and milk whey samples (4μl) were separated through 4% stacking and 12% running PAGE gels at 65Vand 4° C. overnight. Proteins were electrotransferred to nitrocellulose(0.45 μm; Micron Separation) at 200 mA for 1 hour. Membranes were thensequentially blocked with TBS/3% tergitol NP-40, TBS/1% BSA (Sigma), andTBS/0/1% Tween. Membranes were incubated overnight with 1E10⁶ cpm of[¹²⁵I]-IGF-I and IGF-I-BP was visualized by autoradiogroaphy.Radioactivity was quantitated using the Foto/analyst II Visionary Systemand Collage software.

The milk of transgenic sows was found to contain increased levels ofIGFBP5 relative to non-transgenic control animals. No change in serumIGFBP was observed in transgenic sows relative to non-transgeniccontrols.

D. Level of Milk Solids in Transgenic Sows

Total milk solids in transgenic sows and non-transgenic controls wasmeasured by scalding and drying 0.5 g of milk overnight in an oven at100° C. The results are shown in FIG. 6. The level of milk solids waslower in transgenic sows than control sows at day 0 of lactation. Levelswere similar in transgenic and control samples for the remainder of thestudy.

E. Protein Content in the Milk of Transgenic Sows

Protein concentration of milk samples was measured using the BCA assay(Pierce). Results are shown in FIG. 7. Protein levels were higher in themilk of transgenic sows on days 0, and 1 of lactation and were higher incontrol samples on days 8 and 24 of lactation.

F. Lactose Content in the Milk of Transgenic Sows

Lactose content of milk samples was measured using a colorimetric assaybased on the method of Teles (J. Dairy Sci., 61:506 [1978]). The resultsare shown in FIG. 8. The lactose content of milk samples from transgenicand non-transgenic sows was not significantly different.

G. Fat Content in the Milk of Transgenic Sows

Fat content of milk samples was measured using a chloroform/methanolextraction method (Bligh and Dyer, Can. J. Biochem. Phys., 37:911[1959]) that was been modified to a microassay requiring 0.5 ml of milk.The results are shown in FIG. 9. Milk from transgenic sows had a higherfat content than milk from non-transgenic control sows at days 0-2 oflactation.

EXAMPLE 4 Effect of IGF-I Transgenic Milk on Suckling Piglets

A. Weight Gain of Suckling Piglets

The weight of suckling piglets was obtained on days 3, 7, 14, and 21days of lactation. FIG. 10 shows weight gain of piglets sucklingtransgenic and control sows. Piglets suckling transgenic sows showed aslightly increased cumulative weight gain during lactation. On day 24,the mean weight of piglets who nursed from transgenic sows was 5.26±0.15kg (n=50) compared to 5.09±0.15 kg (n=40) in piglets who nursed fromcontrol sows.

B. Lactase Activity of Piglet Intestines

Piglets were killed by electrocution and exsanguination on the indicateddays. The small intestine from the pyloric sphincter to the ileocecalvalve was removed and separated from the mesentery. The weight andlength of the entire intestine was determined and 50 cm samplesrepresenting jejunum (50% total length) and ileum (85% total length)were flushed with ice-cold 0.9% saline. Mucosal samples were collectedby opening each segment longitudinally and scraping the luminal surfacewith a glass slide and then frozen in liquid nitrogen.

Lactase activity in intestinal samples was assayed using previouslydescribed methods (Houle et al., Pediatr. Res., 42:78 [1997]). Briefly,mucosal samples were homogenized in saline containing proteaseinhibitors (1 mM phenylmethy-sulfonylfluoride, 2 mM iodoacetic acid).Lactase activity was determined by incubating intestinal homogenateswith lactose for 60 minutes at 37° C. Liberated glucose was assayed byglucose oxidase. Enzyme activity is expressed as umol glucose/minute/gprotein.

Results are shown in FIG. 11. Lactase activity was higher in intestinalsamples of piglets suckled on transgenic sows relative to control sowson days 4 and 7 of lactation.

EXAMPLE 5 Effect of Exogenous IGF-I on Mammary Growth

The effect of IGF-I overexpression on mammary growth, histomorphology,and nutrient uptake is measured in tissue samples from transgenic andcontrol sows. Samples are collected at different stages of lactation andpost-lactation.

Differences between the transgenic and non-transgenic controls will bedetermined using a repeated measures analysis of variance (Steele andTorrie (eds.), Principles and Procedures of Statistics, Second Edition,McGraw-Hill, New York, N.Y. [1980]). Computations are performed usingthe general linear model procedure in SAS (version 6.04; SAS Institute,Cary, N.C.; SAS, 1985).

A. Histomorphology

Formalin-fixed mammary tissue from sows at different days of lactationis embedded in paraffin, thin-sliced with a microtome, and stained withhematoxylin/eosin. Sections are histologically evaluated for lobularproliferation, secretory activity, and for changes in supporting stromaand adipose tissues (Neuenschwander et al., J. Clin. invest., 97:2225[1996]). Sections showing involutional changes are evaluated forresidual acinar proliferation and secretory change, extent ofducto-lobular luminal secretions, apoptosis of ductolobular epithelialand myoepithelial cells, and the nature and extent of the hostinflammatory response. Apoptosis is identified histologically by thepresence of prominent cytoplasmic eosinophilia, nuclear shrinkages andfragmentation and apoptotic bodies.

B. Apoptosis

The TUNEL assay is used to further localize cells exhibiting DNAfragmentation characteristic of apoptosis (Tanaka et al., 1996). In thisassay, short fragments of DNA formed by endonucleases are detected withthe use of terminal transferase enzyme (an enzyme that attachesdeoxynucleotides to the 3′ end of DNA fragments). Formalin-fixed mammarysections are used. Prepared slides are dipped in paraformaldehyde/PBSand then incubated in proteinase K for 15 minutes at 37° C. The slidesare rinsed in TBS, pH 7.4 and sections are incubated in terminaltransferase buffer (Boeringer Mannhein) containing biotinylated dUTP(Clonetech) in a humid atmosphere at 37° C. for 60 minutes. Endogenousperoxidase activity is inactivated by incubation in H₂O₂ for 20 minutesat room temperature. After washing and blocking with 10% goat serum,slides are incubated with streptavidin-conjugated peroxidase, washedwith TBS, stained with deoxyaminobenzidine for several minutes andcounterstained with methyl green. The number of apoptotic cells iscounted by light microscopy. It is expected that mammary glands of sowsoverexpressing IGF-I will demonstrate decreased apoptosis as compared tocontrols non-transgenic sows.

C. Proliferation

Mammary cells undergoing proliferation will be detectedimmunohistochemically in mammary sections using a monoclonal antibody tocyclin A (Novocastra). Cyclin A is a 60 kDa protein which binds tocyclin-dependent kinase-2 in S to G2 phase of the cell cycle. Cyclin Ais specific to cells undergoing DNA synthesis. Labelled cells aredetected using streptavidin/biotin complex (Vectastain Elite,Novocastra). It is expected that mammary glands of sows overexpressingIGF-I will demonstrate increased proliferation as compared to controlsnon-transgenic sows.

D. Mammary Nutrient Uptake

Mammary amino acid uptake is assayed using well known techniques. Formeasurement of amino acid uptake, biopsy tissue is minced and washedthree times in basal medium (5 mM KCL, 2 mM CaCl₂, 1 mM MgSO₄, 135 mMNaCl, 10 mM glucose, and 10 mM TRIS-BES, 7.4). For measurement ofglucose uptake, concentrations of glucose are varied to determinekinetic properties of glucose transport (Alexander and Carey, [1999]).For determining sodium independence, NaCl in the basal medium isreplaced with 135 mM choline chloride. Kinetic properties of thetransport systems are determined by incubating explant tissue in thepresence of a range of concentrations of lysine, valine, and glucose,plus the respective radiolabeled tracer. After incubation, explants arelightly blotted, weighed, and macromolecules precipitated withtrichloroacetic acid. Soluble radiolabel, representing free amino acidtaken up by the tissue, is counted and uptake is calculated. It isexpected that mammary glands of sows overexpressing IGF-I willdemonstrate increased nutrient uptake as compared to controlsnon-transgenic sows.

EXAMPLE 6 Effect of IGF-I on Piglet Intestinal Development

Piglets are sacrificed and intestinal samples prepared as described inExample 4B above. Samples are analyzed for morphology, digestive, andabsorptive functions as described below.

A. GI Structure and Morphology

DNA and protein content of intestinal samples is determined usingpreviously described methods (Houle et al., Pediatr. Res., 42:78[1997]). Intestinal histomorpholgy is analyzed by embeddingformulin-fixed iejunal samples in paraffin, slicing the samples to 5 μMwith a microtome, and staining with hematoxylin. Villus height, villuswidth, crypt depth, and muscularis thickness are measured using a Nikonmicroscope (Fryer) and Image 1 software (Universal Imaging) in 10vertically well-oriented villi and crypts. Crypt to villus ratios andvillus cross-sectional area will be calculated (Houle et al., Pediatr.Res., 42:78 [1997]). It is expected that mirovillus height will beincreased in piglets suckling transgenic sows as compared to pigletssuckling non-transgenic control sows.

B. Disaccharidase Gene Expression

Lactase and sucrase steady state mRNA abundance is measured usingstandard methods. Total cellular RNA is isolated by the method ofChomczynski (Bio Techniques, 15:532 [1993]), size fractionated byagarose-gel electrophoresis and capillary transferred to nylon membranes(Houle et al., Pediatr. Res., 42:78 [1997]). Blots are hybridized with³²P-dCTP-labeled cDNA probes for lactase (Troelsen et al., J. Biol.Chem., 267:20407 [1992]), sucrase (Chandrasena et al., Cell. Mol. Biol.,38:243 [1992]). RNA expression is quantified using a phosphoimager(Molecular Dynamics) and lactase and sucrase expression is normalized toEF-1α expression. It is expected that disaccharidase activity will beincreased in piglets suckling transgenic sows as compared to pigletssuckling non-transgenic control sows.

C. Nutrient Transport and Ion Secretion

Jejunal and ileal samples are stripped of their muscularis, openedlongitudinally, and mounted in an Ussing chamber apparatus (PhysiologicInstruments). Mucosal and serosal surfaces (0.5 cm²) are exposed to 10mL oxygenated (95%O₂/5% CO₂) Krebs buffer pH 7.4 which is recirculatedfrom a reservoir maintained at 37° C. After a 15 minute equilibration,spontaneous transmural potential difference (mV) and short circuitcurrent are measured (Tappenden et al., 2000). Sodium-dependent nutrienttransport is determined by marking the change in short circuit currentinduced by the addition of glucose and glutamine (10⁻²M) to the mucosalside of the Ussing chamber. Ion secretion is determined by quantifyingthe change in short-circuit current induced by the addition of chloridesecretagogous (10⁻⁴ serotonign and carbachol) to the media bathing theserosal side of the tissue. The Ussing chamber apparatus is connected to4 dual channel voltage/current clamps (VCC MC2, Physiologic Instruments)which each have a computer interface allowing for real time dataacquisition and subsequent analysis (Acquire and Analyze software,Physiologic Instruments). It is expected that Na⁺ coupled glucose andglutamine transport will be increased in piglets suckling transgenicsows as compared to piglets suckling non-transgenic control sows.

D. Nutrient Transporter Gene Expression

Total cellular RNA is isolated and northern blots are prepared asdescribed above (Example 6B). Blots are hybridized with cDNA probes forB⁰ amino acid transporter, SGLT-1, and the Na⁺, K⁺-ATPase α1, andNa⁺,K⁺-ATPase β1 subunits. For GLUT2, blots are hybridized with anantisense riboprobe for GLUT2 generated from plasmid DNA (pGEM-4Z-HTL-3;obtained from G. I. Bell, University of Chicago) and T7 RNA polymerase(Tappenden et al., 1997). Membranes are probed for EF-1α and mRNA isquantified and normalized as described above (Example 6B). It isexpected that Na⁺, K⁺-ATPase gene expression will be increased inpiglets suckling transgenic sows as compared to piglets sucklingnon-transgenic control sows.

E. Nutrient Transporter Protein Abundance

The relative abundance of SGLT-1, GLUT2, and Na⁺,K⁺-ATPase proteins isidentified in isolated brush border and basolateral membranes,respectively, by Western immunoblotting. Brush border and basolateralmembranes are simultaneously isolated as described previously (Tappendenet al., 1998). Briefly, stripped mucosa are homogenized in 2.5 Msucrose-tris buffer and brush border and basolateral membrane fractionsare separated by 3 sequential centrifugation steps. The brush bordermembrane fraction is further purified using a 20% Percoll gradient,followed by calcium chloride precipitation. The basolateral membranefraction is further purified by calcium chloride precipitation. Membranepurity is confirmed through a 10 to 20 fold enrichment in activity ofalkaline phosphatase (brush border) or Na⁺,K⁺-ATPase (basolateral) overthe initial homogenate. Membrane proteins are separated by SDS-PAGE,electroblotted to nitrocellulose and immunoblotted using standardmethods (Tappenden, et al, 1998). Polyclonal (rabbit anti-human) primaryantibodies against GLUT2, SGLT-1 and Na⁺, K⁺-ATPase (Oncogene Science)are used. Bands are detected by chemiluminescence using the SupersignalCL-HRP Substrate System (Pierce) followed by exposure to Hyperfilm ECL(Amersham). Relative protein concentrations are determined bydensitometry. It is expected that Na⁺, K⁺-ATPase, GLUT2, and SGLT-1protein expression will be increased in piglets suckling transgenic sowsas compared to piglets suckling non-transgenic control sows.

EXAMPLE 7 Generation of Alpha-Lactalbumin and IGF-I Transgenic Pigs

A new line of transgenic pigs was generated by mating alpha-lactalbumintransgenic sows to an IGF-I transgenic pig. Piglets were screened andgilts positive for both α-LA/IGF-I (n=7), α-LA alone (n=7), IGF-I alone(n=3) and negative for both transgenes (n=5) were bred to a York boar.Milk yield and piglet growth were assessed throughout lactation. Milkproduction of α-LA (6.56±0.042 kg) and α-LA/IGF-I transgenic (6.51±0.052kg) were higher on day 7 of lactation than IGF-I (5.3±0.062 kg) orcontrol (5.1×0.044 kg) sows. Body weight at weaning tended to be higherin the piglets suckling from sows positive for both transgenes, but thedifference was not statistically significant at this point due to lowsample size. The lines of transgenic swine created using mammaryover-expression of α-LA, IGF-I or both provide means to improve pigproduction.

U.S. Pat. No. 5,850,000 (Bleck, et al., issued Dec. 15, 1998) and U.S.Pat. No. 5,530,177 (Bleck, et al., issued Jun. 25, 1996) are herebyincorporated by reference to the extent not inconsistent with thedisclosure herewith.

All publications and patents mentioned in the above specification areherein incorporated by reference to the extent not inconsistent with thedisclosure herein. Various modifications and variations of the describedmethod and system of the invention will be apparent to those skilled inthe art without departing from the scope and spirit of the invention.Although the invention has been described in connection with specificpreferred embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying outthe invention which are obvious to those skilled in molecular biology,developmental biology, biochemistry, or related fields are intended tobe within the scope of the following claims.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 2 <210> SEQ ID NO 1 <211> LENGTH: 4532<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial      Sequence:alpha-LA/IGF-I gene construct <400> SEQUENCE: 1gatcagtcct gggtggtcat tgaaaggact gatgctgaag ttgaagctcc aa#tactttgg     60ccacctgatg cgaagaactg actcatgtga taagaccctg atactgggaa ag#attgaagg    120caggaggaga agggatgaca gaggatggaa gagttggatg gaatcaccaa ct#cgatggac    180atgagtttga gcaagcttcc aggagttggt aatgggcagg gaagcctggc gt#gctgcagt    240ccatggggtt gcaaagagtt ggacactact gagtgactga actgaactga ta#gtgtaatc    300catggtacag aatataggat aaaaaagagg aagagtttgc cctgattctg aa#gagttgta    360ggatataaaa gtttagaata cctttagttt ggaagtctta aattatttac tt#aggatggg    420tacccactgc aatataagaa atcaggcttt agagactgat gtagagagaa tg#agccctgg    480cataccagaa gctaacagct attggttata gctgttataa ccaatatata ac#caatatat    540tggttatata gcatgaagct tgatgccagc aatttgaagg aaccatttag aa#ctagtatc    600ctaaactcta catgttccag gacactgatc ttaaagctca ggttcagaat ct#tgttttat    660aggctctagg tgtatattgt ggggcttccc tggtggctca gatggtaaag tg#tctgcctg    720caatgtgggt gatctgggtt cgatccctgg cttgggaaga tcccctggag aa#ggaaatgg    780caacccactc tagtactctt acctggaaaa ttccatggac agaggagcct tg#taagctac    840agtccatggg attgcaaaga gttgaacaca actgagcaac taagcacagc ac#agtacagt    900atacacctgt gaggtgaagt gaagtgaagg ttcaatgcag ggtctcctgc at#tgcagaaa    960gattctttac catctgagcc accagggaag cccaagaata ctggagtggg ta#gcctattc   1020cttctccagg ggatcttccc atcccaggaa ttgaactgga gtctcctgca tt#tcaggtgg   1080attcttcacc agctgaacta ccaggtggat actactccaa tattaaagtg ct#taaagtcc   1140agttttccca cctttcccaa aaaggttggg tcactctttt ttaaccttct gt#ggcctact   1200ctgaggctgt ctacaagctt atatatttat gaacacattt attgcaagtt gt#tagtttta   1260gatttacaat gtggtatctg gctatttagt ggtattggtg gttggggatg gg#gaggctga   1320tagcatctca gagggcagct agatactgtc atacacactt ttcaagttct cc#atttttgt   1380gaaatagaaa gtctctggat ctaagttata tgtgattctc agtctctgtg gt#catattct   1440attctactcc tgaccactca acaaggaacc aagatatcaa gggacacttg tt#ttgtttca   1500tgcctgggtt gagtgggcca tgacatatga tgatgtacag tccttttcca ta#ttctgtat   1560gtctctaaga ggaaggagga gttggccgtg gaccctttgt gcattttctg at#tgcttcac   1620ttgtattacc cctgaggccc cctttgttcc tgaaataggt tgggcacatc tt#gcttccta   1680gaaccaacac taccagaaac aacataaata aagccaaatg ggaaacagga tc#atgtttgt   1740aacactcttt gggcaggtaa caatacctag tatggactag agattctggg ga#ggaaagga   1800aaagtggggt gaaattactg aaggaagctc aatgtttctt tgttggtttt ac#tggcctct   1860cttgtcatcc tcttcctgga tgtaaggctt gatgccaggg cccctaaggc tt#tttccaca   1920aataaaagga ggtgagcagt gtggtgaccc catttcagaa tcttgagggg ta#acgaattc   1980taaccaaaat gatgtccttt gtctctctgc tcctggtagg aatcctattc ca#tgccaccc   2040aggctggacc ggagacgctc tgcggggctg agctggtgga tgctcttcag tt#cgtgtgtg   2100gagacagggg attttatttc aacaagccca cagggtatgg atccagcagt cg#gagggcgc   2160cccagacagg catcgtggat gagtgctgct tccggagctg tgatctaagg ag#gctggaga   2220tgtattgcgc acccctaaag cctgccaagt cagcttgata gctcgacgga tc#cccaaaat   2280gtgaggtgtt ccgggagctg aaagacttga agggctacgg aggtgtcagt tt#gcctgaat   2340gtgagttccc tgctattttg ctttgtccca taattcatcc tcttcactct tt#ccctccat   2400tctcttcatc ctcttttccc cctctacttt taattatcaa acaattctct ta#tttgttta   2460ctcttttatt acatttattt atctgcctct cctttttccc attgtctgat cc#tttggaac   2520tcttttcacc ttaacaagat actctgtggt ctgccatatt tggagattgg tt#ggagagcc   2580tttttcggtc tgggaataca ggtcctcatt tatgctatac atgaacatcc tt#gtgaaatc   2640tctttttcgt ctttctttca ggggtctgta ccgcgtttca taccagtggt ta#tgacacac   2700aagccatagt acaaaacaat gacagcacag aatatggact cttccagata aa#taataaaa   2760tttggtgcaa agacgaccag aaccctcact caagcaacat ctgtaacatc tc#ctgtgaca   2820gtgagtaact tctttttact ctgttcctgt gtttttctga aacctactcc tg#ggataacc   2880tccttttttt tggtgtgaag cacacctctg gcttcactgc cttggactcc aa#attaactg   2940tgggacttga taataccgag taagaggctc ttagaatttt tcattaacac ta#aatcccca   3000gacagtttct taaagttcct gggtaggtga cctgagctgt ttggggatct tg#atgtataa   3060taccctgtat tttcagacta agttggttga tgaagttgat aattcctaag ga#gctgcccc   3120agagaagaga agggagtcct tacctaggga taggcattac tgtattaaat tt#ctcaccca   3180gaaggcaaca ggcataagcc tctagttcag agaaaaccag agaagaggga aa#ttcattat   3240ccttctgggt aatacttagc tctctcattt tttccaccag aggctcctgc ca#gagttcct   3300ggatgatgat cttactgatg acattatgtg tgtcaagaag attctggata aa#gtaggaat   3360taactactgg tgagtcacct ctctattttt cacttaatct ttcctctctt tc#ttctcagt   3420cctttcgtcc cagcactata ctcctttctc tctatttctt ggtcttttaa gc#tagaatgt   3480aatcttaaaa acaaaaatca tcaagcagac tccggtttcc aattttgaag ct#tcacttac   3540ttcactcccg ttagcaattt tcctacctaa gggtccctaa tagagggctg ag#atccagga   3600tttccttcac caggacttga acatctaatt ctacttgttc agtcctacat cc#taaggcac   3660gccctttgac cactgccccg caattttctt ggagttttaa aaaatggacc tt#actccact   3720aagtggctca gtgtctctag ccatgtggct aggaaagtct gtctgtaatt tt#aacccaca   3780gtcttccacc tcagccttcc tggggataaa gctagatgta aatctaacca ag#atcctgtc   3840agtaatttgc cttgtctcct tcttcatgat caggttggcc cataaagcac tc#tgttctga   3900gaagctggat cagtggctct gtgagaagtt gtgaacacct gctgtctttg ct#gcttctgt   3960cctctttctg ttcctggaac tcctctgccc cgtggctacc tcgttttgct tc#tttgtacc   4020cccttgaagc taactcgtct ctgagccctg ggccctgtag tgacaatgga ca#tgtaagga   4080ctaatctcca ggtgtgcatg aatggcgctc tggacttttg acccttgctc ga#tgtccctg   4140atggcgcttt taatgcaaca gtacatattc cacttttgtc ccgaataaaa ag#cctgattt   4200tgagtggctg gctgtatttt cttcctggtg ggagagggag gaaatagggt ga#gtaggtag   4260acctggccat gggtcacaga ccccttcatc tctactaaag aggatagaga gg#ctgaactt   4320ataacaactc aaagatggag attactttct gtattaattc aattcaacag ag#ttttattg   4380atcacctagc ataatttaaa gagctatgga ggggatctaa agttgactaa aa#gcatctct   4440tacctaaact gctgctaagt cacttcagtt gtgtccgact ctgtgtgacc cc#atagacgg   4500 tagcccacaa ggctcccatg tccctggaat tc       #                   #        4532 <210> SEQ ID NO 2 <211> LENGTH: 210<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:IGF-I<400> SEQUENCE: 2ggaccggaga cgctctgcgg ggctgagctg gtggatgctc ttcagttcgt gt#gtggagac     60aggggatttt atttcaacaa gcccacaggg tatggatcca gcagtcggag gg#cgccccag    120acaggcatcg tggatgagtg ctgcttccgg agctgtgatc taaggaggct gg#agatgtat    180 tgcgcacccc taaagcctgc caagtcagct         #                   #          210

What is claimed is:
 1. A transgenic ungulate having a genome, saidgenome comprising a heterologous nucleic acid sequence encoding a growthfactor operably linked to a mammary preferential promoter, whereindescendants of said transgenic ungulate express an increased amount ofgrowth factor in their milk as compared to control non-transgenicungulates.
 2. The transgenic ungulate of claim 1, wherein said growthfactor is selected from the group consisting of insulin-like growthfactor I, insulin-like growth factor II, epidermal growth factor,platelet-derived growth factor, fibroblast growth factor, andtransforming growth factor.
 3. The transgenic ungulate of claim 2,wherein said insulin-like growth factor I is selected from the groupconsisting of human, porcine, and bovine insulin-like growth factor I.4. The transgenic ungulate claim 2, wherein said insulin-like growthfactor I comprises SEQ ID NO:2.
 5. The transgenic ungulate of claim 1,wherein said mammary preferential promoter is an alpha-lactalbuminpromoter.
 6. The transgenic ungulate of claim 1, wherein said mammarypreferential promoter is selected from the group consisting of bovine,porcine, and human mammary preferential promoters.
 7. The transgenicungulate of claim 1, wherein said transgenic ungulate is a pig.
 8. Thetransgenic ungulate of claim 1, wherein said transgenic ungulate isselected from the group consisting of a male transgenic ungulate and afemale transgenic ungulate.
 9. The transgenic ungulate of claim 1,wherein gametes of said transgenic ungulate comprise said heterologousnucleic acid sequence.
 10. The transgenic ungulate claim 1, wherein saidheterologous nucleic acid sequence is encoded by SEQ ID NO:
 1. 11. Acomposition comprising milk from a transgenic ungulate having a genome,said genome comprising a heterologous nucleic acid sequence encoding agrowth factor operably linked to a mammary preferential promoter,wherein said milk comprises an increased amount of growth factor ascompared to milk from control non-transgenic ungulates.
 12. Thecomposition of claim 11, wherein said growth factor is selected from thegroup consisting of insulin-like growth factor I, insulin-like growthfactor II, epidermal growth factor, platelet-derived growth factor,fibroblast growth factor, and transforming growth factor.
 13. Thecomposition of claim 12, wherein said insulin-like growth factor I isselected from the group consisting of human, porcine, and bovineinsulin-like growth factor I.
 14. The composition of claim 12, whereinsaid insulin-like growth factor I comprises SEQ ID NO:2.
 15. Thecomposition of claim 12, wherein said milk is obtained from a pig.
 16. Amethod of increasing weight gain in a suckling ungulate offspringcomprising: a) providing i) a transgenic ungulate having a genome, saidgenome comprising a heterologous nucleic acid sequence encoding a growthfactor gene operably linked to a mammary preferential promoter, whereinsaid transgenic ungulate expresses an increased amount of growth factorin its milk as compared to control non-transgenic ungulates; and ii) asuckling ungulate offspring; and b) providing said suckling ungulateoffspring milk of said transgenic ungulate, wherein said sucklingungulate offspring has increased weight gain relative to a sucklingungulate offspring provided milk of a non-transgenic ungulate.
 17. Themethod of claim 16, wherein said growth factor is selected from thegroup consisting of insulin-Like growth factor I, insulin-like growthfactor II, epidermal growth factor, platelet-derived growth factor,fibroblast growth factor, and transforming growth factor.
 18. The methodof claim 17, wherein said insulin-like growth factor I is selected fromthe group consisting of human, porcine, and bovine insulin-like growthfactor I.
 19. The method of claim 17, wherein said insulin-like growthfactor comprises SEQ ID NO:2.
 20. The method of claim 16, wherein saidmammary preferential promoter is an alpha-lactalbumin promoter.
 21. Themethod of claim 16, wherein said mammary specific promoter is selectedfrom the group consisting of human, porcine, and bovine mammary specificpromoter.
 22. The method of claim 16, wherein said transgenic ungulateis a pig.
 23. The method of claim 16, wherein said transgenic ungulateis selected from the group consisting of a male transgenic ungulate anda female transgenic ungulate.
 24. The method of claim 16, wherein saidsuckling ungulate offspring is a piglet.
 25. The method of claim 16,wherein gametes of said transgenic ungulate comprise said heterologousnucleic acid sequence.
 26. The method of claim 16, wherein saidheterologous nucleic acid sequence is encoded by SEQ ID NO:
 1. 27. Amethod of increasing intestinal lactase activity in a suckling ungulateoffspring, comprising: a) providing i) a transgenic ungulate having agenome, said genome comprising a heterologous nucleic acid sequenceencoding a growth factor operably linked to a mammary preferentialpromoter, wherein said transgenic ungulate expresses an increased amountof growth factor in its milk as compared to control non-transgenicungulates; and ii) a suckling ungulate offspring; and b) providing saidsuckling ungulate offspring milk of said transgenic ungulate, whereinsaid suckling ungulate offspring has increased intestinal lactaseactivity relative to a suckling ungulate offspring provided milk of anon-transgenic ungulate.
 28. The method of claim 27, wherein said growthfactor is selected from the group consisting of insulin-like growthfactor I, insulin-like growth factor II, epidermal growth factor,platelet-derived growth factor, fibroblast growth factor, andtransforming growth factor.
 29. The method of claim 28, wherein saidinsulin-like growth factor I is selected from the group consisting ofhuman, porcine, and bovine insulin-like growth factor I.
 30. The methodof claim 28, wherein said insulin-like growth factor comprises SEQ IDNO:2.
 31. The method of claim 27, wherein said mammary preferentialpromoter is an alpha-lactalbumin promoter.
 32. The method of claim 27,wherein said mammary specific promoter is selected from the groupconsisting of human, porcine, and bovine mammary specific promoter. 33.The method of claim 27, wherein said transgenic ungulate is a pig. 34.The method of claim 27, wherein said transgenic ungulate is selectedfrom the group consisting of a male transgenic ungulate and a femaletransgenic ungulate.
 35. The method of claim 27, wherein said sucklingungulate offspring is a piglet.
 36. The method of claim 27, whereingametes of said transgenic ungulate comprise said heterologous nucleicacid sequence.
 37. The method of claim 27, wherein said heterologousnucleic acid sequence is encoded by SEQ ID NO:
 1. 38. A method ofincreasing intestinal cell division in a suckling ungulate offspring,comprising: a) providing i) a transgenic ungulate having a genome, saidgenome comprising a heterologous nucleic acid sequence encoding a growthfactor operably linked to a mammary preferential promoter, wherein saidtransgenic ungulate expresses an increased amount of growth factor inits milk as compared to control non-transgenic ungulates; and ii) asuckling ungulate offspring; and b) providing said suckling ungulateoffspring milk of said transgenic ungulate, wherein said sucklingungulate offspring has increased intestinal cell division relative to asuckling ungulate offspring provided milk of a non-transgenic ungulate.39. The method of claim 38, wherein said growth factor is selected fromthe group consisting of insulin-like growth factor I, insulin-likegrowth factor II, epidermal growth factor, platelet-derived growthfactor, fibroblast growth factor, and transforming growth factor. 40.The method of claim 39, wherein said insulin-like growth factor I isselected from the group consisting of human, porcine, and bovineinsulin-like growth factor I.
 41. The method of claim 39, wherein saidinsulin-like growth factor comprises SEQ ID NO:2.
 42. The method ofclaim 38, wherein said mammary preferential promoter is analpha-lactalbumin promoter.
 43. The method of claim 38, wherein saidmammary preferential promoter is selected from the group consisting ofhuman, porcine, and bovine mammary preferential promoter.
 44. The methodof claim 38, wherein said transgenic ungulate is a pig.
 45. The methodof claim 38, wherein said transgenic ungulate is selected from the groupconsisting of a male transgenic ungulate and a female transgenicungulate.
 46. The method of claim 38, wherein said suckling ungulateoffspring is a piglet.
 47. The method of claim 38, wherein gametes ofsaid transgenic ungulate comprise said heterologous nucleic acidsequence.
 48. The method of claim 38, wherein said heterologous nucleicacid sequence is encoded by SEQ ID NO:
 1. 49. A method of increasingintestinal villi length in a suckling ungulate offspring, comprising: a)providing i) a transgenic ungulate having a genome, said genomecomprising a heterologous nucleic acid sequence encoding a growth factoroperably linked to a mammary preferential promoter, wherein saidtransgenic ungulate expresses an increased amount of growth factor inits milk as compared to control non-transgenic ungulates; and ii) asuckling ungulate offspring; and b) providing said suckling ungulateoffspring milk of said transgenic ungulate, wherein said sucklingungulate offspring has increased intestinal villi length relative to asuckling ungulate offspring provided milk of a non-transgenic ungulate.50. The method of claim 49, wherein said growth factor is selected fromthe group consisting of insulin-like growth factor I, insulin-likegrowth factor II, epidermal growth factor, platelet-derived growthfactor, fibroblast growth factor, and transforming growth factor. 51.The method of claim 50, wherein said insulin-like growth factor I isselected from the group consisting of human, porcine, and bovineinsulin-like growth factor I.
 52. The method of claim 50, wherein saidinsulin-like growth factor comprises SEQ ID NO:2.
 53. The method ofclaim 49, wherein said mammary preferential promoter is analpha-lactalbumin promoter.
 54. The method of claim 49, wherein saidmammary preferential promoter is selected from the group consisting ofhuman, porcine, and bovine mammary preferential promoter.
 55. The methodof claim 49, wherein said transgenic ungulate is a pig.
 56. The methodof claim 49, wherein said transgenic ungulate is selected from the groupconsisting of a male transgenic ungulate and a female transgenicungulate.
 57. The method of claim 49, wherein said suckling ungulateoffspring is a piglet.
 58. The method of claim 49, wherein gametes ofsaid transgenic ungulate comprise said heterologous nucleic acidsequence.
 59. The method of claim 49, wherein said heterologous nucleicacid sequence is encoded by SEQ ID NO:
 1. 60. A method of increasingresistance to intestinal pathogens in a suckling ungulate offspring,comprising: a) providing i) a transgenic ungulate having a genome, saidgenome comprising a heterologous nucleic acid sequence encoding a growthfactor operably linked to a mammary preferential promoter; wherein saidtransgenic ungulate expresses an increased amount of growth factor inits milk as compared to control non-transgenic ungulates; and ii) asuckling ungulate offspring; and b) providing said suckling ungulateoffspring milk of said transgenic ungulate, wherein said sucklingungulate offspring has increased resistance to intestinal parasitesrelative to a suckling ungulate offspring provided milk of anon-transgenic ungulate.
 61. The method of claim 60, wherein said growthfactor is selected from the group consisting of insulin-like growthfactor I, insulin-like growth factor II, epidermal growth factor,platelet-derived growth factor, fibroblast growth factor, andtransforming growth factor.
 62. The method of claim 61, wherein saidinsulin-like growth factor I is selected from the group consisting ofhuman, porcine, and bovine insulin-like growth factor I.
 63. The methodof claim 61, wherein said insulin-like growth factor comprises SEQ IDNO:2.
 64. The method of claim 60, wherein said mammary preferentialpromoter is an alpha-lactalbumin promoter.
 65. The method of claim 60,wherein said mammary preferential promoter is selected from the groupconsisting of human, porcine, and bovine mammary preferential promoter.66. The method of claim 60, wherein said transgenic ungulate is a pig.67. The method of claim 60, wherein said transgenic ungulate is selectedfrom the group consisting of a male transgenic ungulate and a femaletransgenic ungulate.
 68. The method of claim 60, wherein said sucklingungulate offspring is a piglet.
 69. The method of claim 60, whereingametes of said transgenic ungulate comprise said heterologous nucleicacid sequence.
 70. The method of claim 60, wherein said heterologousnucleic acid sequence is encoded by SEQ ID NO:
 1. 71. The method ofclaim 60, wherein said intestinal pathogen is selected from the groupconsisting of rotovirus, coronavirus, E. coli, and Salmonella.